Cryogenic and LOX Based Propulsion Systems for Robotic Planetary Missions

National Research Council Canada - National Science Library

Valentian, Dominique

2005-01-01

Robotic planetary missions use almost exclusively storable propellants. However, it is clear that the use LOX/LH2 and LOX/HC combinations will offer a tremendous payload gain for most robotic missions...

In vivo formation of beta-oxidized metabolites of leukotriene E4 in the rat

International Nuclear Information System (INIS)

Perrin, P.; Zirrolli, J.; Stene, D.O.; Lellouche, J.P.; Beaucourt, J.P.; Murphy, R.C.

1989-01-01

Intraperitoneal administration of [ 3 H]-leukotriene E4 in the rat resulted in the appearance of radiolabel in urine and feces. Separation of polar urinary metabolites and chromatographic comparison of synthetic metabolites indicated the in vivo formation of omega-oxidized metabolites of LTE4 with sequential beta-oxidation. Furthermore, the metabolite identified as 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl-LTE4 substantiates the biochemical pathway of beta-oxidation in vivo involving the 2,4-dienoyl CoA reductase as an integral step. These results substantiate beta-oxidation of sulfidopeptide leukotrienes in vivo and these metabolites account for some of the major urinary metabolites of this class of lipid mediator

Total hydrocarbon content (THC) testing in liquid oxygen (LOX) systems

Science.gov (United States)

Meneghelli, B. J.; Obregon, R. E.; Ross, H. R.; Hebert, B. J.; Sass, J. P.; Dirschka, G. E.

2015-12-01

The measured Total Hydrocarbon Content (THC) levels in liquid oxygen (LOX) systems at Stennis Space Center (SSC) have shown wide variations. Examples of these variations include the following: 1) differences between vendor-supplied THC values and those obtained using standard SSC analysis procedures; and 2) increasing THC values over time at an active SSC test stand in both storage and run vessels. A detailed analysis of LOX sampling techniques, analytical instrumentation, and sampling procedures will be presented. Additional data obtained on LOX system operations and LOX delivery trailer THC values during the past 12-24 months will also be discussed. Field test results showing THC levels and the distribution of the THC's in the test stand run tank, modified for THC analysis via dip tubes, will be presented.

[Spectral analysis of polyphenol oxidase (PPO) and lipoxygenase (LOX) treated by pulsed electric field].

Science.gov (United States)

Luo, Wei; Zhang, Ruo-Bing; Chen, Jie; Wang, Li-Ming; Guan, Zhi-Cheng; Jia, Zhi-Dong

2009-08-01

Inactivation effect of pulsed electric field (PEF) on polyphenol oxidase (PPO) and lipoxygenase (LOX) was investigated using a laboratory PEF system with a coaxial treatment chamber. Circular dichroism (CD) and fluorescence analysis were used to study the conformation change of the protein. The experimental results show that PPO and LOX can be effectively inactivated by the PEF treatment. Inactivation effect of PPO and LOX increases with the increase in the applied electric strength and the treatment time. Activity of PPO and LOX can be reduced by 60.3% and 21.7% at 20 kV x cm(-1) after being treated for 320 micros respectively. The decrease of the negative peaks (208 and 215 nm in PPO spectra, 208 nm and 218 nm in LOX spectra) in CD spectra of PPO and LOX shows that PEF treatment caused a loss of alpha-helix and increase in beta-sheet content, indicating that conformation changes occur in the secondary structure of PPO and LOX enzyme. This effect was strengthened as the applied electric field increased: alpha-helical content of PPO and LOX was 56% and 29% after being treated at 8 kV x cm(-1), however, when the electric field was increased up to 20 kV x cm(-1), alpha-helical content of PPO and LOX decreased to 21% and 16% respectively. The decrease rate of alpha-helix and increase rate of beta-sheet in PPO are higher than LOX, indicating that the second conformation of PPO is less resistant to PEF treatment than LOX. The fluorescence intensity of LOX increases after PEF treatment. At the same time, increasing the applied pulsed electric field increases the fluorescence intensity emitted. Fluorescence measurements confirm that tertiary conformation changes occur in the local structure of LOX. However the possible mechanism of the conformation change induced by the PEF treatment is beyond the scope of the present investigation.

Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

International Nuclear Information System (INIS)

Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

2013-01-01

Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis

Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

Energy Technology Data Exchange (ETDEWEB)

Lee, Ha Young, E-mail: hayoung@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Sang Doo [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Baek, Suk-Hwan [Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Choi, Joon Hyuk [Department of Pathology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Cho, Kyung-Hyun [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Zabel, Brian A. [Palo Alto Institute for Research and Education, Veterans Affairs Hospital, Palo Alto, CA 94304 (United States); Bae, Yoe-Sik, E-mail: yoesik@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of)

2013-03-29

Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

Characterization of the lipoxygenase (LOX) gene family in the Chinese white pear (Pyrus bretschneideri) and comparison with other members of the Rosaceae.

Science.gov (United States)

Li, Meng; Li, Leiting; Dunwell, Jim M; Qiao, Xin; Liu, Xing; Zhang, Shaoling

2014-06-07

Lipoxygenases (LOXs), a type of non-haem iron-containing dioxygenase, are ubiquitous enzymes in plants and participate in the formation of fruit aroma which is a very important aspect of fruit quality. Amongst the various aroma volatiles, saturated and unsaturated alcohols and aldehydes provide the characteristic aroma of the fruit. These compounds are formed from unsaturated fatty acids through oxidation, pyrolysis and reduction steps. This biosynthetic pathway involves at least four enzymes, including LOX, the enzyme responsible for lipid oxidation. Although some studies have been conducted on the LOX gene family in several species including Arabidopsis, soybean, cucumber and apple, there is no information from pear; and the evolutionary history of this gene family in the Rosaceae is still not resolved. In this study we identified 107 LOX homologous genes from five Rosaceous species (Pyrus bretschneideri, Malus × domestica, Fragaria vesca, Prunus mume and Prunus persica); 23 of these sequences were from pear. By using structure analysis, phylogenic analysis and collinearity analysis, we identified variation in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Expression of certain pear LOX genes during fruit development was verified by analysis of transcriptome data. 23 LOX genes were identified in pear and these genes were found to have undergone a duplication 30-45 MYA; most of these 23 genes are functional. Specific gene duplication was found on chromosome4 in the pear genome. Useful information was provided for future research on the evolutionary history and transgenic research on LOX genes.

Metabolite kinetics: formation of acetaminophen from deuterated and nondeuterated phenacetin and acetanilide on acetaminophen sulfation kinetics in the perfused rat liver preparation

International Nuclear Information System (INIS)

Pang, K.S.; Waller, L.; Horning, M.G.; Chan, K.K.

1982-01-01

The role of hepatic intrinsic clearance for metabolite formation from various precursors on subsequent metabolite elimination was was investigated in the once-through perfused rat liver preparation. Two pairs of acetaminophen precursors: [ 14 C] phenacetin-d5 and [ 3 H] phenacetin-do, [ 14 C] acetanilide and [ 3 H] phenacetin were delivered by constant flow (10 ml/min/liver) either by normal or retrograde perfusion to the rat liver preparations. The extents of acetaminophen sulfation were compared within the same preparation. The data showed that the higher the hepatocellular activity (intrinsic clearance) for acetaminophen formation, the greater the extent of subsequent acetaminophen sulfation. The findings were explained on the basis of blood transit time and metabolite duration time. Because of blood having only a finite transit time in liver, the longer the drug requires for metabolite formation, the less time will remain for metabolite sulfation and the less will be the degree of subsequent sulfation. Conversely, when the drug forms the primary metabolite rapidly, a longer time will remain for the metabolite to be sulfated in liver to result in a greater degree of metabolite sulfation. Finally, the effects of hepatic intrinsic clearances for metabolite formation and zonal distribution of enzyme systems for metabolite formation and elimination in liver are discussed

On mechanisms of reactive metabolite formation from drugs.

Science.gov (United States)

Claesson, Alf; Spjuth, Ola

2013-04-01

Idiosyncratic adverse drug reactions (IADRs) cause a broad range of clinically severe conditions of which drug induced liver injury (DILI) in particular is one of the most frequent causes of safety-related drug withdrawals. The underlying cause is almost invariably formation of reactive metabolites (RM) which by attacking macromolecules induc eorgan injuries. Attempts are being made in the pharmaceutical industry to lower the risk of selecting unfit compounds as clinical candidates. Approaches vary but do not seem to be overly successful at the initial design/synthesis stage. We review here the most frequent categories of mechanisms for RM formation and propose that many cases of RMs encountered within early ADME screening can be foreseen by applying chemical and metabolic knowledge. We also mention a web tool, SpotRM, which can be used for efficient look-up and learning about drugs that have recognized IADRs likely caused by RM formation.

Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments.

Science.gov (United States)

Felker, A; Mosimann, C

2016-01-01

Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments. In addition, advanced transgenesis technologies such as phiC31 or CRISPR-Cas9-based knock-ins are even further promoting zebrafish transgenesis for Cre/lox applications. In this chapter, we will first introduce the basics of Cre/lox methodology, CreERT2 regulation by tamoxifen, as well as the utility of Tol2 and other contemporary transgenesis techniques for Cre/lox experiments. We will then outline in detail practical experimental steps for efficient transgenesis toward the creation of single-insertion transgenes and will introduce protocols for 4-hydroxytamoxifen-mediated CreERT2 induction to perform spatiotemporal lox transgene regulation experiments in zebrafish embryos. Last, we will discuss advanced experimental applications of Cre/lox beyond traditional lineage tracing approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

The LOX-1 Scavenger Receptor and Its Implications in the Treatment of Vascular Disease

Directory of Open Access Journals (Sweden)

M. W Twigg

2012-01-01

Full Text Available Cardiovascular disease is the leading cause of death. The disease is due to atherosclerosis which is characterized by lipid and fat accumulation in arterial blood vessel walls. A key causative event is the accumulation of oxidised low density lipoprotein particles within vascular cells, and this is mediated by scavenger receptors. One such molecule is the LOX-1 scavenger receptor that is expressed on endothelial, vascular smooth muscle, and lymphoid cells including macrophages. LOX-1 interaction with OxLDL particles stimulates atherosclerosis. LOX-1 mediates OxLDL endocytosis via a clathrin-independent internalization pathway. Transgenic animal model studies show that LOX-1 plays a significant role in atherosclerotic plaque initiation and progression. Administration of LOX-1 antibodies in cellular and animal models suggest that such intervention inhibits atherosclerosis. Antiatherogenic strategies that target LOX-1 function using gene therapy or small molecule inhibitors would be new ways to address the increasing incidence of vascular disease in many countries.

Insulin-like growth factor I reduces lipid oxidation and foam cell formation via downregulation of 12/15-lipoxygenase.

Science.gov (United States)

Sukhanov, Sergiy; Snarski, Patricia; Vaughn, Charlotte; Lobelle-Rich, Patricia; Kim, Catherine; Higashi, Yusuke; Shai, Shaw-Yung; Delafontaine, Patrice

2015-02-01

We have shown that insulin-like growth factor I (IGF-1) infusion in Apoe(-/-) mice decreased atherosclerotic plaque size and plaque macrophage and lipid content suggesting that IGF-1 suppressed formation of macrophage-derived foam cells. Since 12/15-lipoxygenase (12/15-LOX) plays an important role in OxLDL and foam cell formation, we hypothesized that IGF-1 downregulates 12/15-LOX, thereby suppressing lipid oxidation and foam cell formation. We found that IGF-1 decreased 12/15-LOX plaque immunopositivity and serum OxLDL levels in Apoe(-/-) mice. IGF-1 reduced 12/15-LOX protein and mRNA levels in cultured THP-1 macrophages and IGF-1 also decreased expression of STAT6 transcription factor. IGF-1 reduction in macrophage 12/15-LOX was mediated in part via a PI3 kinase- and STAT6-dependent transcriptional mechanism. IGF-1 suppressed THP-1 macrophage ability to oxidize lipids and form foam cells. IGF-1 downregulated 12/15-LOX in human blood-derived primary macrophages and IGF-1 decreased LDL oxidation induced by these cells. IGF-1 reduced LDL oxidation and formation of foam cells by wild type murine peritoneal macrophages, however these effects were completely blocked in 12/15-LOX-null macrophages suggesting that the ability of IGF-1 to reduce LDL oxidation and foam cells formation is dependent on its ability to downregulate 12/15-LOX. Overall our data demonstrate that IGF-1 reduces lipid oxidation and foam cell formation via downregulation of 12/15-LOX and this mechanism may play a major role in the anti-atherosclerotic effects of IGF-1. Published by Elsevier Ireland Ltd.

Insulin-like Growth Factor I Reduces Lipid Oxidation and Foam Cell Formation via Downregulation of 12/15-lipoxygenase

Science.gov (United States)

Sukhanov, Sergiy; Snarski, Patricia; Vaughn, Charlotte; Lobelle-Rich, Patricia; Kim, Catherine; Higashi, Yusuke; Shai, Shaw-Yung; Delafontaine, Patrice

2014-01-01

Objective We have shown that insulin-like growth factor I (IGF-1) infusion in Apoe−/− mice decreased atherosclerotic plaque size and plaque macrophage and lipid content suggesting that IGF-1 suppressed formation of macrophage-derived foam cells. Since 12/15-lipoxygenase (12/15-LOX) plays an important role in OxLDL and foam cell formation, we hypothesized that IGF-1 downregulates 12/15-LOX, thereby suppressing lipid oxidation and foam cell formation. Approach and Results We found that IGF-1 decreased 12/15-LOX plaque immunopositivity and serum OxLDL levels in Apoe−/− mice. IGF-1 reduced 12/15-LOX protein and mRNA levels in cultured THP-1 macrophages and IGF-1 also decreased expression of STAT6 transcription factor. IGF-1 reduction in macrophage 12/15-LOX was mediated in part via a PI3 kinase- and STAT6-dependent transcriptional mechanism. IGF-1 suppressed THP-1 macrophage ability to oxidize lipids and form foam cells. IGF-1 downregulated 12/15-LOX in human blood-derived primary macrophages and IGF-1 decreased LDL oxidation induced by these cells. IGF-1 reduced LDL oxidation and formation of foam cells by wild type murine peritoneal macrophages, however these effects were completely blocked in 12/15-LOX-null macrophages suggesting that the ability of IGF-1 to reduce LDL oxidation and foam cells formation is dependent on its ability to downregulate 12/15-LOX. Conclusions Overall our data demonstrate that IGF-1 reduces lipid oxidation and foam cell formation via downregulation of 12/15-LOX and this mechanism may play a major role in the anti-atherosclerotic effects of IGF-1. PMID:25549319

LOX: Inferring level of expression from diverse methods of census sequencing

KAUST Repository

Zhang, Zhang

2010-06-10

Summary: We present LOX (Level Of eXpression) that estimates the Level Of gene eXpression from high-throughput-expressed sequence datasets with multiple treatments or samples. Unlike most analyses, LOX incorporates a gene bias model that facilitates integration of diverse transcriptomic sequencing data that arises when transcriptomic data have been produced using diverse experimental methodologies. LOX integrates overall sequence count tallies normalized by total expressed sequence count to provide expression levels for each gene relative to all treatments as well as Bayesian credible intervals. © The Author 2010. Published by Oxford University Press. All rights reserved.

LOX: Inferring level of expression from diverse methods of census sequencing

KAUST Repository

Zhang, Zhang; Ló pez-Girá ldez, Francesc Francisco; Townsend, Jeffrey P.

2010-01-01

Summary: We present LOX (Level Of eXpression) that estimates the Level Of gene eXpression from high-throughput-expressed sequence datasets with multiple treatments or samples. Unlike most analyses, LOX incorporates a gene bias model that facilitates integration of diverse transcriptomic sequencing data that arises when transcriptomic data have been produced using diverse experimental methodologies. LOX integrates overall sequence count tallies normalized by total expressed sequence count to provide expression levels for each gene relative to all treatments as well as Bayesian credible intervals. © The Author 2010. Published by Oxford University Press. All rights reserved.

LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis

DEFF Research Database (Denmark)

Cox, Thomas R; Bird, Demelza; Baker, Ann-Marie

2013-01-01

of metastastic tumor cells. We show that LOX-dependent collagen crosslinking is involved in creating a growth-permissive fibrotic microenvironment capable of supporting metastatic growth by enhancing tumor cell persistence and survival. We show that therapeutic targeting of LOX abrogates not only the extent...... to which fibrosis manifests, but also prevents fibrosis-enhanced metastatic colonization. Finally, we show that the LOX-mediated collagen crosslinking directly increases tumor cell proliferation, enhancing metastatic colonization and growth manifesting in vivo as increased metastasis. This is the first...... time that crosslinking of collagen I has been shown to enhance metastatic growth. These findings provide an important link between ECM homeostasis, fibrosis, and cancer with important clinical implications for both the treatment of fibrotic disease and cancer....

Design, synthesis, and biological evaluation of prenylated chalcones as 5-LOX inhibitors.

Science.gov (United States)

Reddy, Nimmanapalli P; Aparoy, Polamarasetty; Reddy, T Chandra Mohan; Achari, Chandrani; Sridhar, P Ramu; Reddanna, Pallu

2010-08-15

Ten novel mono- and di-O-prenylated chalcone derivatives were designed on the basis of a homology derived molecular model of 5-lipoxygenase (5-LOX). The compounds were docked into 5-LOX active site and the binding characteristics were quantified using LUDI. To verify our theoretical assumption, the molecules were synthesized and tested for their 5-LOX inhibitory activities. The synthesis was carried out by Claisen-Schmidt condensation reaction of mono- and di-O-prenylated acetophenones with appropriate aldehydes. 5-LOX in vitro inhibition assay showed higher potency of di-O-prenylated chalcones than their mono-O-prenylated chalcone analogs. Compound 5e exhibited good inhibition with an IC(50) at 4 microM. The overall trend for the binding energies calculated and LUDI score was in good qualitative agreement with the experimental data. Further, the compound 5e showed potent anti-proliferative effects (GI(50) at 9 microM) on breast cancer cell line, MCF-7. Copyright 2010 Elsevier Ltd. All rights reserved.

Lipoxygenase independent hexanal formation in isolated soy proteins induced by reducing agents.

Science.gov (United States)

Lei, Q; Boatright, W L

2008-08-01

Compared to corresponding controls, 6.5 mM dithiothreitol (DTT) elevated headspace hexanal level over aqueous slurries of both commercial isolated soy proteins (ISP) and laboratory ISP prepared with 80 degrees C treatment. Further analysis revealed that lipoxygenase (LOX) activity was not detected from these ISP, indicating that LOX is not involved in the observed hexanal increase. Levels of the induced headspace hexanal over the ISP aqueous slurries were proportional to the amount of DTT added in the range of 0 to 65 mM. Subsequent systematic investigations with model systems revealed that iron was required for the reducing agent-induced hexanal formation from linoleic acid. Erythorbate, another reducing agent, can also induce hexanal formation in both ISP and model systems. As a comparison, the LOX activity and hexanal synthesis in defatted soy flour were examined. The corresponding results showed that defatted soy flour maintained high LOX activities and that hexanal synthesis in such sample was significantly inhibited by high concentration DTT (above 130 mM). Data from the current investigation demonstrate the existence of LOX independent hexanal formation induced by reducing agents in ISP and the potential requirement of iron as a catalyst.

Eliminating LH2 in LOX-collect space launchers - Key to on-demand capability

Science.gov (United States)

Leingang, J. L.; Carreiro, L. R.; Maurice, L. Q.

1993-01-01

Two air-breathing reusable two-stage space launch vehicle concepts are proposed, in which the first stage employs turboramjet propulsion and the second stage uses rockets, which are expected to provide very rapid response launch of 10,000 lb polar-orbit payloads. In both concepts, liquid oxygen (LOX) for the second stage is collected during first stage ascent, thus eliminating the need for LOX ground servicing facilities. In the first concept, liquid hydrogen in the amount just sufficient to condense and collect second state LOX is the only cryogenic fluid that is loaded on the vehicle at takeoff. The second concept uses the heat sink of conventional jet propulsion fuel and water coolant to drive a lightweight adaptation of the commercial LOX production process, eliminating all cryogenics at takeoff. Both concepts should permit true launch-on-demand capability with aircraftlike ground operations.

Efecto in vitro del D-002 sobre la actividad enzimática de la 5-lipoxigenasa (5-LOX Effect of D-002 on 5-lipooxygenase (5-LOX enzyme activity in vitro

Directory of Open Access Journals (Sweden)

Yohani Pérez Guerra

2012-06-01

enzyme activity in vitro by using the cytosolic preparations from rat liver homogenates. Methods: testing conditions were as follows: cytosolic fraction (50 µg of protein dissolved in 0.2 mol/L borate buffer solution (pH 9 and linoleic acid (7.8-250 mmol/L as substrate. Parallel samples were incubated with Tween-20/H2O (2 % only (vehicle, control samples, D-002 (0.9-1 000 µg/mL or Lyprinol (500 µg/mL (reference substance. The enzyme activity, evaluated through the formation of conjugated dienes, was assessed by the absorbance changes at 234 nm (5-lox measured in a UV-visible spectrophotometer. Results: the in vitro addition of D-002 produced a significant, dose-dependent (r=0.980; p< 0.001 (IC50= 95.34 µg/mL and uncompetitive inhibition of 5-LOX activity, whose maximal inhibition (70 % was achieved with 500 µg/mL. Conclusions: thisstudy demonstrates that D-002 effectively inhibits 5-LOX enzymatic activity, an effect that may partially explain the anti-inflammatory effects of D-002 in experimental models in vivo.

Activated human mast cells induce LOX-1-specific scavenger receptor expression in human monocyte-derived macrophages.

Directory of Open Access Journals (Sweden)

Mervi Alanne-Kinnunen

Full Text Available Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs.Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1 mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α, and transforming growth factor beta (TGF-β1, which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell -induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.

Induced sclerotium formation exposes new bioactive metabolites from Aspergillus sclerotiicarbonarius

DEFF Research Database (Denmark)

Petersen, Lene Maj; Frisvad, Jens Christian; Knudsen, Peter Boldsen

2015-01-01

Sclerotia are known to be fungal survival structures, and induction of sclerotia may prompt production of otherwise undiscovered metabolites. Aspergillus sclerotiicarbonarius (IBT 28362) was investigated under sclerotium producing conditions, which revealed a highly altered metabolic profile. Four...... new compounds were isolated from cultivation under sclerotium formation conditions and their structures elucidated using different analytical techniques (HRMS, UV, 1D and 2D NMR). This included sclerolizine, an alkylated and oxidized pyrrolizine, the new emindole analog emindole SC and two new...

The impact of selected strains of probiotic bacteria on metabolite formation in set yoghurt

NARCIS (Netherlands)

Settachaimongkon, S.; Nout, M.J.R.; Antunes Fernandes, E.C.; Hooijdonk, van A.C.M.; Zwietering, M.H.; Smid, E.J.; Valenberg, van H.J.F.

2014-01-01

The influence of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 in cofermentation with traditional starters on metabolite formation in set yoghurt was evaluated. Microbial activity during fermentation and refrigerated storage was investigated by monitoring bacterial

The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes.

Science.gov (United States)

Johänning, Janina; Kröner, Patrick; Thomas, Maria; Zanger, Ulrich M; Nörenberg, Astrid; Eichelbaum, Michel; Schwab, Matthias; Brauch, Hiltrud; Schroth, Werner; Mürdter, Thomas E

2018-03-01

Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug-drug-interactions.

Numerical analysis of flow and heat transfer in the VAFB LOX storage Dewar tank

Science.gov (United States)

Tam, L. T.; Singhal, A. K.

1984-01-01

The present report describes numerical simulation of three-dimensional transient distributions of velocity and temperature of liquid oxygen (LOX) in the LOX Dewar tank of Vendenberg Air Force Base (VAFB). The present analyses cover the replenish time period only. Four test cases have been considered. For all four cases, the input boundary conditions are comprised of LOX facility heat loads, drain flow rates, recirculation flow rates and dewar heating. All the quantities are prescribed as functions of time. The first two test cases considered sensitivity of results to the computational grid. In Case 3, system heat load was changed, while in Case 4, a lower LOX level was specified. Cases 1 and 2 showed that the temperatures were not sensitive to the grid refinement. This provided a basic check on the numerical model. Cases 3 and 4 showed that the thermal boundary layer motion near the tank surface becomes more significant at the late time, e.g., 5 1/2 hours from replenish start. Comparison between results of Cases 3 and 4 showed, as expected, that the smaller initial LOX volume given in Case 4, results in higher temperature level. All calculated velocity and temperature distributions were found to be plausible.

Anomalous baroreflex functionality inherent in floxed and Cre-Lox mice: an overlooked physiological phenotype.

Science.gov (United States)

Tsai, Ching-Yi; Poon, Yan-Yuen; Chen, Chang-Han; Chan, Samuel H H

2017-10-01

The last two decades have seen the emergence of Cre-Lox recombination as one of the most powerful and versatile technologies for cell-specific genetic engineering of mammalian cells. Understandably, the primary concerns in the practice of Cre-Lox recombination are whether the predicted genome has been correctly modified and the targeted phenotypes expressed. Rarely are the physiological conditions of the animals routinely examined because the general assumption is that they are normal. Based on corroborative results from radiotelemetric recording, power spectral analysis, and magnetic resonance imaging/diffusion tensor imaging in brain-derived neurotrophic factor-floxed mice, the present study revealed that this assumption requires amendment. We found that despite comparable blood pressure and heart rate with C57BL/6 or Cre mice under the conscious state, floxed and Cre-Lox mice exhibited diminished baroreflex-mediated sympathetic vasomotor tone and cardiac vagal baroreflex. We further found that the capacity and plasticity of baroreflex of these two strains of mice under isoflurane anesthesia were retarded, as reflected by reduced connectivity between the nucleus tractus solitarii and rostral ventrolateral medulla or nucleus ambiguus. The identification of anomalous baroreflex functionality inherent in floxed and Cre-Lox mice points to the importance of incorporating physiological phenotypes into studies that engage gene manipulations such as Cre-Lox recombination. NEW & NOTEWORTHY We established that anomalous baroreflex functionality is inherent in floxed and Cre-Lox mice. These two mouse strains exhibited diminished baroreflex-mediated sympathetic vasomotor tone and cardiac vagal baroreflex under the conscious state, retarded capacity and plasticity of baroreflex under isoflurane anesthesia, and reduced connectivity between key nuclei in the baroreflex neural circuits. Copyright © 2017 the American Physiological Society.

Combustion chamber heat transfer characterization of LOX/hydrocarbon-type propellants

Science.gov (United States)

Schoenman, Leonard

1987-01-01

The gas-side heat transfer rates for LOX/propane and LOX/ethanol are experimentally characterized using a 1000 lb thrust water-cooled calorimeter chamber. The effects of injector element type and fuel film cooling are defined as a function of mixture ratio. The interaction of fuel injected through the resonator cavities on heat transfer and wall soot buildup are displayed as a function of time, axial distance, fuel coolant flow rate, and mixture ratio. Comparisons between clean-burning ethanol and sooting propane show a large difference between the two fuels and significantly higher than expected heat flux levels for ethanol in the throat region.

15-Lipoxygenase metabolites of α-linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome

Science.gov (United States)

Kumar, Naresh; Gupta, Geetika; Anilkumar, Kotha; Fatima, Naireen; Karnati, Roy; Reddy, Gorla Venkateswara; Giri, Priyanka Voori; Reddanna, Pallu

2016-01-01

The ratio of ω-6 to ω-3 polyunsaturated fatty acids (PUFAs) appears to be critical in the regulation of various pathophysiological processes and to maintain cellular homeostasis. While a high proportion of dietary intake of ω-6 PUFAs is associated with various inflammatory disorders, higher intake of ω-3 PUFAs is known to offer protection. It is now well established that beneficial effects of ω-3 PUFAs are mediated in part by their oxygenated metabolites mainly via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. However, the down-stream signaling pathways that are involved in these anti-inflammatory effects of ω-3 PUFAs have not been elucidated. The present study evaluates the effects of 15-LOX metabolites of α-linolenic acid (ALA, ω-3 PUFA) on lipopolysaccharide (LPS) induced inflammation in RAW 264.7 cells and peritoneal macrophages. Further, the effect of these metabolites on the survival of BALB/c mice in LPS mediated septic shock and also polymicrobial sepsis in Cecal Ligation and Puncture (CLP) mouse model was studied. These studies reveal the anti-inflammatory effects of 13-(S)-hydroperoxyoctadecatrienoic acid [13-(S)-HPOTrE] and 13-(S)-hydroxyoctadecatrienoic acid [13-(S)-HOTrE] by inactivating NLRP3 inflammasome complex through the PPAR-γ pathway. Additionally, both metabolites also deactivated autophagy and induced apoptosis. In mediating all these effects 13-(S)-HPOTrE was more potent than 13-(S)-HOTrE. PMID:27535180

Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt

NARCIS (Netherlands)

Settachaimongkon, S.; Valenberg, van H.J.F.; Winata, V.; Wang, X.; Nout, M.J.R.; Hooijdonk, van A.C.M.; Zwietering, M.H.; Smid, E.J.

2015-01-01

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk,

The protective effect of bergamot oil extract on lecitine-like oxyLDL receptor-1 expression in balloon injury-related neointima formation.

Science.gov (United States)

Mollace, Vincenzo; Ragusa, Salvatore; Sacco, Iolanda; Muscoli, Carolina; Sculco, Francesca; Visalli, Valeria; Palma, Ernesto; Muscoli, Saverio; Mondello, Luigi; Dugo, Paola; Rotiroti, Domenicantonio; Romeo, Francesco

2008-06-01

Lectin-like oxyLDL receptor-1 (LOX-1) has recently been suggested to be involved in smooth muscle cell (SMC) proliferation and neointima formation in injured blood vessels. This study evaluates the effect of the nonvolatile fraction (NVF), the antioxidant component of bergamot essential oil (BEO), on LOX-1 expression and free radical generation in a model of rat angioplasty. Common carotid arteries injured by balloon angioplasty were removed after 14 days for histopathological, biochemical, and immunohistochemical studies. Balloon injury led to a significant restenosis with SMC proliferation and neointima formation, accompanied by increased expression of LOX-1 receptor, malondialdehyde and superoxide formation, and nitrotyrosine staining. Pretreatment of rats with BEO-NVF reduced the neointima proliferation together with free radical formation and LOX-1 expression in a dose-dependent manner. These results suggest that natural antioxidants may be relevant in the treatment of vascular disorders in which proliferation of SMCs and oxyLDL-related endothelial cell dysfunction are involved.

Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.

Science.gov (United States)

Döhlemann, Johannes; Brennecke, Meike; Becker, Anke

2016-09-10

The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti. The hsdMSR operon encodes a type-I restriction-modification (R-M) system. Transformation of S. meliloti is counteracted by the restriction endonuclease HsdR degrading DNA which lacks the appropriate methylation pattern. We provide a stable S. meliloti hsdR deletion mutant showing enhanced transformation with Escherichia coli-derived plasmid DNA and demonstrate that using an E. coli plasmid donor, expressing S. meliloti methyl transferase genes, is an alternative strategy of increasing the transformation efficiency of S. meliloti. Furthermore, we devise a novel cloning-free genome editing (CFGE) method for S. meliloti, Agrobacterium tumefaciens and Xanthomonas campestris, and demonstrate the applicability of this method for intricate applications of the Cre/lox recombination system in S. meliloti. An enhanced Cre/lox system, allowing for serial deletions of large genomic regions, was established. An assay of lox spacer mutants identified a set of lox sites mediating specific recombination. The availability of several non-promiscuous Cre recognition sites enables simultaneous specific Cre/lox recombination events. CFGE combined with Cre/lox recombination is put forward as powerful approach for targeted genome editing, involving serial steps of manipulation to expedite the genetic accessibility of S. meliloti as chassis. Copyright © 2016 Elsevier B.V. All rights reserved.

Formation and Change of Chloroplast-Located Plant Metabolites in Response to Light Conditions

Directory of Open Access Journals (Sweden)

Yiyong Chen

2018-02-01

Full Text Available Photosynthesis is the central energy conversion process for plant metabolism and occurs within mature chloroplasts. Chloroplasts are also the site of various metabolic reactions involving amino acids, lipids, starch, and sulfur, as well as where the production of some hormones takes place. Light is one of the most important environmental factors, acting as an essential energy source for plants, but also as an external signal influencing their growth and development. Plants experience large fluctuations in the intensity and spectral quality of light, and many attempts have been made to improve or modify plant metabolites by treating them with different light qualities (artificial lighting or intensities. In this review, we discuss how changes in light intensity and wavelength affect the formation of chloroplast-located metabolites in plants.

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

Directory of Open Access Journals (Sweden)

Yaíma L Lightfoot

Full Text Available Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA depleted βLox5 cells, or βLox5 ρ(0 cells. βLox5 ρ(0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

The rationale for targeting the LOX family in cancer

DEFF Research Database (Denmark)

Barker, Holly E; Cox, Thomas R; Erler, Janine T

2012-01-01

The therapeutic targeting of extracellular proteins is becoming hugely attractive in light of evidence implicating the tumour microenvironment as pivotal in all aspects of tumour initiation and progression. Members of the lysyl oxidase (LOX) family of proteins are secreted by tumours and are the ......The therapeutic targeting of extracellular proteins is becoming hugely attractive in light of evidence implicating the tumour microenvironment as pivotal in all aspects of tumour initiation and progression. Members of the lysyl oxidase (LOX) family of proteins are secreted by tumours...... and are the subject of much effort to understand their roles in cancer. In this Review we discuss the roles of members of this family in the remodelling of the tumour microenvironment and their paradoxical roles in tumorigenesis and metastasis. We also discuss how targeting this family of proteins might lead to a new...... avenue of cancer therapeutics....

Summary of LOX/CH4 Thruster Technology Development at NASA/MSFC

Science.gov (United States)

Greene, Sandra Elam

2015-01-01

In recent years, a variety of injectors for liquid oxygen (LOX) and methane (CH4) propellant systems have been designed, fabricated, and demonstrated with hot-fire testing at Marshall Space Flight Center (MSFC). Successful designs for liquid methane (LCH4) and gaseous methane (GCH4) have been developed. A variety of chambers, including a transpiration cooled design, along with uncooled ablatives and refractory metals, have also been hot-fire tested by MSFC for use with LOX/LCH4 injectors. Hot-fire testing has also demonstrated multiple ignition source options. Heat flux data for selected injectors has been gathered by testing with a calorimeter chamber. High performance and stable combustion have been demonstrated, along with designs for thrust levels ranging from 500 to 7,000 lbf. The newest LOX/CH4 injector and chamber developed by MSFC have been fabricated with additive manufacturing techniques and include unique design features to investigate regenerative cooling with methane. This low cost and versatile hardware offers a design for 4,000 lbf thrust and will be hot-fire tested at MSFC in 2015. Its design and operation can easily be scaled for use in systems with thrust levels up to 25,000 lbf.

Isolation and characterization of wild-type lipoxygenase LOX(Psa)1 from Pleurotus sapidus.

Science.gov (United States)

Plagemann, Ina; Krings, Ulrich; Berger, Ralf G

2014-01-01

The lipoxygenase LOX(Psa) 1 of Pleurotus sapidus, originally investigated because of its ability to oxidize (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was isolated from the peptidase-rich lyophilisate using a three-step purification scheme including preparative isoelectric focusing and chromatographic techniques. Nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS) of the purified enzyme and peptide mass fingerprint analysis gave 38 peptides of the lipoxygenase from P. sapidus. Nearly 50% of the 643 amino acids long sequence encoded by the cDNA was covered. Both terminal peptides of the native LOX(Psa) 1 were identified by de novo sequencing, and the postulated molecular mass of 72.5 kDa was confirmed. With linoleic acid as the substrate, the LOX(Psa)1 showed a specific activity of 113 U mg(-1) and maximal activity at pH 7.0 and 30 degrees C, respectively.

Design of a 2000 lbf LOX/LCH4 Throttleable Rocket Engine for a Vertical Lander

Science.gov (United States)

Lopez, Israel

Liquid oxygen (LOX) and liquid methane (LCH4) has been recognized as an attractive rocket propellant combination because of its in-situ resource utilization (ISRU) capabilities, namely in Mars. ISRU would allow launch vehicles to carry greater payloads and promote missions to Mars. This has led to an increasing interest to develop spacecraft technologies that employ this propellant combination. The UTEP Center for Space Exploration and Technology Research (cSETR) has focused part of its research efforts to developing LOX/LCH4 systems. One of those projects includes the development of a vertical takeoff and landing vehicle called JANUS. This vehicle will employ a LOX/LCH 4 propulsion system. The main propulsion engine is called CROME-X and is currently being developed as part of this project. This rocket engine will employ LOX/LCH4 propellants and is intended to operate from 2000-500 lbf thrust range. This thesis describes the design and development of CROME-X. Specifically, it describes the design process for the main engine components, the design criteria for each, and plans for future engine development.

Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

Energy Technology Data Exchange (ETDEWEB)

Dong, Q.; Xiang, R.; Zhang, D.Y.; Qin, S. [Department of Cardiology, The First Affiliated Hospital, Chongqing Medical University, Chongqing (China)

2013-09-19

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.

Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Directory of Open Access Journals (Sweden)

Ren Wang

2008-06-01

Full Text Available Lipoxygenase (LOX, EC1.13.11.12 is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+ and transformed into Escherichia coli BL21 (DE3. Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG and the purified recombinant protein was obtained by His·Bind® Kits. Further assay showed that the purified recombinant protein exhibited the LOX activity. The optimum pH was 4.8 (acetate buffer and the optimum temperature was 30°C for the above enzyme. Thus, the recombinant might confer an available usage for the synthesis of jasmonate in vitro, and also provides a possibility for elucidating the inter-relationship between the primary structure of the plant seed lipoxygenase protein and its physiological functions.

Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

International Nuclear Information System (INIS)

Dong, Q.; Xiang, R.; Zhang, D.Y.; Qin, S.

2013-01-01

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression

Optimized Jasmonic Acid Production by Lasiodiplodia theobromae Reveals Formation of Valuable Plant Secondary Metabolites.

Directory of Open Access Journals (Sweden)

Felipe Eng

Full Text Available Jasmonic acid is a plant hormone that can be produced by the fungus Lasiodiplodia theobromae via submerged fermentation. From a biotechnological perspective jasmonic acid is a valuable feedstock as its derivatives serve as important ingredients in different cosmetic products and in the future it may be used for pharmaceutical applications. The objective of this work was to improve the production of jasmonic acid by L. theobromae strain 2334. We observed that jasmonic acid formation is dependent on the culture volume. Moreover, cultures grown in medium containing potassium nitrate as nitrogen source produced higher amounts of jasmonic acid than analogous cultures supplemented with ammonium nitrate. When cultivated under optimal conditions for jasmonic acid production, L. theobromae secreted several secondary metabolites known from plants into the medium. Among those we found 3-oxo-2-(pent-2-enyl-cyclopentane-1-butanoic acid (OPC-4 and hydroxy-jasmonic acid derivatives, respectively, suggesting that fungal jasmonate metabolism may involve similar reaction steps as that of plants. To characterize fungal growth and jasmonic acid-formation, we established a mathematical model describing both processes. This model may form the basis of industrial upscaling attempts. Importantly, it showed that jasmonic acid-formation is not associated to fungal growth. Therefore, this finding suggests that jasmonic acid, despite its enormous amount being produced upon fungal development, serves merely as secondary metabolite.

Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

Directory of Open Access Journals (Sweden)

Himanshu Singh

Full Text Available Cellular metabolite analyses by (13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13C-labelled acetate ((13CH(3-COOH or CH(3-(13COOH supported that both the (13C nuclei give rise to bicarbonate and CO2(aq. The observed metabolite(s upon further incubation led to the production of starch and triacylglycerol (TAG in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

Vinpocetine attenuates lipid accumulation and atherosclerosis formation

International Nuclear Information System (INIS)

Cai, Yujun; Li, Jian-Dong; Yan, Chen

2013-01-01

Highlights: •Vinpocetine attenuates hyperlipidemia-induced atherosclerosis in a mouse model. •Vinpocetine antagonizes ox-LDL uptake and accumulation in macrophages. •Vinpocetine blocks the induction of ox-LDL receptor LOX-1 in vitro and in vivo. -- Abstract: Atherosclerosis, the major cause of myocardial infarction and stroke, is a chronic arterial disease characterized by lipid deposition and inflammation in the vessel wall. Cholesterol, in low-density lipoprotein (LDL), plays a critical role in the pathogenesis of atherosclerosis. Vinpocetine, a derivative of the alkaloid vincamine, has long been used as a cerebral blood flow enhancer for treating cognitive impairment. Recent study indicated that vinpocetine is a potent anti-inflammatory agent. However, its role in the pathogenesis of atherosclerosis remains unexplored. In the present study, we show that vinpocetine significantly reduced atherosclerotic lesion formation in ApoE knockout mice fed with a high-fat diet. In cultured murine macrophage RAW264.7 cells, vinpocetine markedly attenuated oxidized LDL (ox-LDL) uptake and foam cell formation. Moreover, vinpocetine greatly blocked the induction of ox-LDL receptor 1 (LOX-1) in cultured macrophages as well as in the LOX-1 level in atherosclerotic lesions. Taken together, our data reveal a novel role of vinpocetine in reduction of pathogenesis of atherosclerosis, at least partially through suppressing LOX-1 signaling pathway. Given the excellent safety profile of vinpocetine, this study suggests vinpocetine may be a therapeutic candidate for treating atherosclerosis

Vinpocetine attenuates lipid accumulation and atherosclerosis formation

Energy Technology Data Exchange (ETDEWEB)

Cai, Yujun [Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642 (United States); Li, Jian-Dong [Center for Inflammation, Immunity and Infection, and Department of Biology, Georgia State University, Atlanta, GA 30303 (United States); Yan, Chen, E-mail: Chen_Yan@urmc.rochester.edu [Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642 (United States)

2013-05-10

Highlights: •Vinpocetine attenuates hyperlipidemia-induced atherosclerosis in a mouse model. •Vinpocetine antagonizes ox-LDL uptake and accumulation in macrophages. •Vinpocetine blocks the induction of ox-LDL receptor LOX-1 in vitro and in vivo. -- Abstract: Atherosclerosis, the major cause of myocardial infarction and stroke, is a chronic arterial disease characterized by lipid deposition and inflammation in the vessel wall. Cholesterol, in low-density lipoprotein (LDL), plays a critical role in the pathogenesis of atherosclerosis. Vinpocetine, a derivative of the alkaloid vincamine, has long been used as a cerebral blood flow enhancer for treating cognitive impairment. Recent study indicated that vinpocetine is a potent anti-inflammatory agent. However, its role in the pathogenesis of atherosclerosis remains unexplored. In the present study, we show that vinpocetine significantly reduced atherosclerotic lesion formation in ApoE knockout mice fed with a high-fat diet. In cultured murine macrophage RAW264.7 cells, vinpocetine markedly attenuated oxidized LDL (ox-LDL) uptake and foam cell formation. Moreover, vinpocetine greatly blocked the induction of ox-LDL receptor 1 (LOX-1) in cultured macrophages as well as in the LOX-1 level in atherosclerotic lesions. Taken together, our data reveal a novel role of vinpocetine in reduction of pathogenesis of atherosclerosis, at least partially through suppressing LOX-1 signaling pathway. Given the excellent safety profile of vinpocetine, this study suggests vinpocetine may be a therapeutic candidate for treating atherosclerosis.

Abscisic Acid Induced Changes in Production of Primary and Secondary Metabolites, Photosynthetic Capacity, Antioxidant Capability, Antioxidant Enzymes and Lipoxygenase Inhibitory Activity of Orthosiphon stamineus Benth.

Directory of Open Access Journals (Sweden)

Mohd Hafiz Ibrahim

2013-07-01

Full Text Available An experiment was conducted to investigate and distinguish the relationships in the production of total phenolics, total flavonoids, soluble sugars, H2O2, O2−, phenylalanine ammonia lyase (PAL activity, leaf gas exchange, antioxidant activity, antioxidant enzyme activity [ascorbate peroxidase (APX, catalase (CAT, superoxide dismutase (SOD and Lipoxygenase inhibitory activity (LOX] under four levels of foliar abscisic acid (ABA application (0, 2, 4, 6 µM for 15 weeks in Orthosiphon stamineus Benth. It was found that the production of plant secondary metabolites, soluble sugars, antioxidant activity, PAL activity and LOX inhibitory activity was influenced by foliar application of ABA. As the concentration of ABA was increased from 0 to 6 µM the production of total phenolics, flavonoids, sucrose, H2O2, O2−, PAL activity and LOX inhibitory activity was enhanced. It was also observed that the antioxidant capabilities (DPPH and ORAC were increased. This was followed by increases in production of antioxidant enzymes APX, CAT and SOD. Under high application rates of ABA the net photosynthesis and stomatal conductance was found to be reduced. The production of primary and secondary metabolites displayed a significant positive relationship with H2O2 (total phenolics, r2 = 0.877; total flavonoids, r2 = 0.812; p ≤ 0.05 and O2− (total phenolics, r2 = 0.778; total flavonoids, r2 = 0.912; p ≤ 0.05. This indicated that increased oxidative stress at high application rates of ABA, improved the production of phytochemicals.

LOX/LH2 vane pump for auxiliary propulsion systems

Science.gov (United States)

Hemminger, J. A.; Ulbricht, T. E.

1985-01-01

Positive displacement pumps offer potential efficiency advantages over centrifugal pumps for future low thrust space missions. Low flow rate applications, such as space station auxiliary propulsion or dedicated low thrust orbiter transfer vehicles, are typical of missions where low flow and high head rise challenge centrifugal pumps. The positive displacement vane pump for pumping of LOX and LH2 is investigated. This effort has included: (1) a testing program in which pump performance was investigated for differing pump clearances and for differing pump materials while pumping LN2, LOX, and LH2; and (2) an analysis effort, in which a comprehensive pump performance analysis computer code was developed and exercised. An overview of the theoretical framework of the performance analysis computer code is presented, along with a summary of analysis results. Experimental results are presented for pump operating in liquid nitrogen. Included are data on the effects on pump performance of pump clearance, speed, and pressure rise. Pump suction performance is also presented.

Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination.

Science.gov (United States)

Betz, U A; Vosshenrich, C A; Rajewsky, K; Müller, W

1996-10-01

The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments. We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage. The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already

Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System.

Science.gov (United States)

Kasai, Yuki; Harayama, Shigeaki

2016-01-01

The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after

Size does matter: Cre-mediated somatic deletion efficiency depends on the distance between the target lox-sites

NARCIS (Netherlands)

Coppoolse, E.R.; Vroomen, de M.J.; Gennip, van F.; Hersmus, B.J.M.; Haaren, van M.J.

2005-01-01

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were

A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco

NARCIS (Netherlands)

Mlynarova, L.; Nap, J.P.H.

2003-01-01

To study the impact of different DNA configurations on the stability of transgene expression, a variant of the cre gene was developed. This variant allows for the highly efficient in planta removal of its own loxP-flanked coding sequence as well as other DNAs flanked by ectopic heterospecific lox

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

Science.gov (United States)

Nam, Bo Young; Kim, Dong Ki; Park, Jung Tak; Kang, Hye-Young; Paeng, Jisun; Kim, Seonghun; Park, Jimin; Um, Jae Eun; Oh, Hyung Jung; Han, Seung Hyeok; Yoo, Tae-Hyun; Kang, Shin-Wook

2015-12-15

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species. Copyright © 2015 the American Physiological Society.

Liquid oxygen liquid acquisition device bubble point tests with high pressure lox at elevated temperatures

Science.gov (United States)

Jurns, J. M.; Hartwig, J. W.

2012-04-01

When transferring propellant in space, it is most efficient to transfer single phase liquid from a propellant tank to an engine. In earth's gravity field or under acceleration, propellant transfer is fairly simple. However, in low gravity, withdrawing single-phase fluid becomes a challenge. A variety of propellant management devices (PMDs) are used to ensure single-phase flow. One type of PMD, a liquid acquisition device (LAD) takes advantage of capillary flow and surface tension to acquire liquid. The present work reports on testing with liquid oxygen (LOX) at elevated pressures (and thus temperatures) (maximum pressure 1724 kPa and maximum temperature 122 K) as part of NASA's continuing cryogenic LAD development program. These tests evaluate LAD performance for LOX stored in higher pressure vessels that may be used in propellant systems using pressure fed engines. Test data shows a significant drop in LAD bubble point values at higher liquid temperatures, consistent with lower liquid surface tension at those temperatures. Test data also indicates that there are no first order effects of helium solubility in LOX on LAD bubble point prediction. Test results here extend the range of data for LOX fluid conditions, and provide insight into factors affecting predicting LAD bubble point pressures.

Risk Assessment and scaling for the SLS LOx ET

Science.gov (United States)

Osipov, Viatcheslav V.; Hafiychuk, Halyna; Devine, Ekaterina V.; Khasin, Michael; Smelyanskiy, Vadim N.

2012-01-01

In this report we analyze the transpiration cooling by He bubble injection of the long LOx tank feedline heated by the environment heat. We consider possible hazards that can arise in the proposed design of the SLS core stage where the feedline length is much longer than that used in the Space Shuttle.

Effect of Omega-3 Fatty Acid Supplementation on Oxylipins in a Routine Clinical Setting

Directory of Open Access Journals (Sweden)

Christoph Schmöcker

2018-01-01

Full Text Available Omega-6 polyunsaturated fatty acid (n-6 PUFA is the predominant polyunsaturated fatty acid (PUFA, especially in Western diet. A high omega-6/omega-3 ratio in Western diets is implicated in the development of cardiovascular diseases and inflammatory processes. Studies in animal models and in humans have demonstrated beneficial effects of omega-3 PUFA (n-3 PUFA in a variety of diseases, including cardiac arrhythmias and inflammatory diseases, as well as breast and colon cancer. The molecular mechanisms underlying the effects of n-3 PUFA are still not well understood. Possible mechanisms include competition between n-3 and n-6 PUFAs at the cyclooxygenase (COX and lipoxygenase (LOX and cytochrome P450 levels, and subsequent formation of oxylipins with specific anti-inflammatory or anti-arrhythmic effects. In this study, we report the impact of routine long-term treatment with prescription-grade n-3 PUFA (either 840 mg or 1680 mg per day on blood cell membrane fatty acid composition, as well as plasma oxylipin patterns, in a patient population with severe hyperlipidemia and cardiovascular disease who are on standard lipid-lowering and cardioprotective medications. Lipidomics analyses were performed by LC/ESI-MS/MS. Supplementation led to a dose-dependent increase in n-3 PUFA eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA in the blood cell fraction. We also observed a dose-dependent increase in EPA- and DHA-derived epoxy metabolites, whereas the effect of n-3 PUFA supplementation on LOX-dependent EPA- and DHA-derived hydroxy metabolites was less pronounced, with a tendency towards lower metabolites in subjects with higher n-3 PUFA levels. These data thus generally confirm effects of n-3 PUFA supplementation observed previously in healthy individuals. Additionally, they indicate a suppressive effect of high n-3 PUFA supplementation on the formation of LOX metabolites in the context of concomitant aspirin medication.

α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

Directory of Open Access Journals (Sweden)

Yuan-Fei Peng

Full Text Available RNA interference (RNAi has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1 was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3 and non-HCC cell lines (L-02, Hela and SW1116 were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5 was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.The AFP-miRNA system could silence target gene (Beclin 1 but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1 in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA was successfully established. The system provides a usable tool for HCC-specific RNAi

The 9-lipoxygenase Osr9-LOX1 interacts with the 13-lipoxygenase-mediated pathway to regulate resistance to chewing and piercing-sucking herbivores in rice.

Science.gov (United States)

Zhou, Guoxin; Ren, Nan; Qi, Jingfeng; Lu, Jing; Xiang, Caiyu; Ju, Hongping; Cheng, Jiaan; Lou, Yonggen

2014-09-01

Oxylipins produced by the 13-lipoxygenase (LOX) have been reported to play an important role in plant defense responses to herbivores. Yet, the role of oxylipins produced by the 9-LOX pathway in this process remains largely unknown. Here we cloned a gene encoding a chloroplast-localized 9-LOX, Osr9-LOX1, from rice. Transcriptional analysis revealed that herbivore infestation, mechanical wounding and jasmonic acid (JA) treatment either repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but did at later stages, whereas salicylic acid (SA) treatment quickly increased the transcript level of Osr9-LOX1. Antisense expression of Osr9-lox1 (as-r9lox1) decreased the amount of wound-induced (Z)-3-hexenal but increased levels of striped stem borer (SSB)-induced linolenic acid, JA, SA and trypsin protease inhibitors. These changes were associated with increased resistance in rice to the larvae of the SSB Chilo suppressalis. In contrast, although no significant differences were observed in the duration of the nymph stage or the number of eggs laid by female adults between the brown planthopper (BPH) Nilaparvata lugens that fed on as-r9lox1 lines and BPH that fed on wild-type (WT) rice plants, the survival rate of BPH nymphs that fed on as-r9lox1 lines was higher than that of nymphs that fed on WT plants, possibly because of a higher JA level. The results demonstrate that Osr9-LOX1 plays an important role in regulating an herbivore-induced JA burst and cross-talk between JA and SA, and in controlling resistance in rice to chewing and phloem-feeding herbivores. © 2014 Scandinavian Plant Physiology Society.

Formation of glutathione conjugates by reactive metabolites of vinylidene chloride in microsomes and isolated hepatocytes

International Nuclear Information System (INIS)

Liebler, D.C.; Meredith, M.J.; Guengerich, F.P.

1985-01-01

Oxidation of the vinyl halide carcinogen and hepatotoxin vinylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, 2-chloroacetic acid, and 1,1-dichloroethylene oxide. The roles of these metabolites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyl chloride reacted with model thiols at least 10(3)-fold faster than did 1,1-dichloroethylene oxide and at least 10(5)-fold faster than did 2,2-dichloroacetaldehyde or 2-chloroacetic acid. Microsomal covalent binding of [ 14 C]VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichloro-1-hydroxy)ethylglutathione, 2-(S-glutathionyl)acetate, and S-(2-glutathionyl)acetylglutathione, a novel conjugate containing both stable (thioether) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyl)acetate were released into the incubation medium. Both 2-(S-glutathionyl)acetate and S-(2-glutathionyl)acetylglutathione were formed with [ 35 S]GSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyl)-acetylglutathione underwent carbonyl substitution with added [ 35 S]GSH, suggesting that this conjugate may participate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC metabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols

Growth of Pseudomonas sp. TX1 on a wide range of octylphenol polyethoxylate concentrations and the formation of dicarboxylated metabolites.

Science.gov (United States)

Lin, Yi-Wen; Guo, Gia-Luen; Hsieh, Hsiao-Cheng; Huang, Shir-Ly

2010-04-01

Pseudomonas sp. TX1, is able to use octylphenol polyethoxylates (OPEO(n), or Triton X-100; average n = 9.5) as a sole carbon source. It can grow on 0.05-20% of OPEO(n) with a specific growth rate of 0.34-0.44 h(-1). High-performance liquid chromatography-mass spectrometer analysis of OPEO(n) degraded metabolites revealed that strain TX1 was able to shorten the ethoxylate chain and produce octylphenol (OP). Furthermore, formation of the short carboxylate metabolites, such as carboxyoctylphenol polyethoxylates (COPEO(n), n = 2, 3) and carboxyoctylphenol polyethoxycarboxylates (COPEC(n), n = 2, 3) began at the log stage, while octylphenol polyethoxycarboxylates (OPEC(n), n = 1-3) was formed at the stationary phase. All the short-ethoxylated metabolites, OPEO(n), OPEC(n), COPEO(n), and COPEC(n), accumulated when the cells were in the stationary phase. This study is the first to demonstrate the formation of COPEO(n) and COPEC(n) from OPEO(n) by an aerobic bacterium. Copyright 2009 Elsevier Ltd. All rights reserved.

Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting….

Science.gov (United States)

Tsien, Joe Z

2016-01-01

Defining and manipulating specific neurons in the brain has garnered enormous interest in recent years, because such an approach is now widely recognized as crucial for deepening our understanding of how the brain works. When I started exploring the Cre-loxP recombination for brain research in the early 1990s, it was written off as a dead-end project by a young fool. Yet over the past 20 years, Cre-lox recombination-mediated neurogenetics has emerged as one of the most powerful and versatile technology platforms for cell-specific gene knockouts, transgenic overexpression, Brainbow imaging, neural pathway tracing with retrovirus and CLARITY, chemical genetics, and optogenetics. Its popularity and greater utility in neuroscience research is also largely thanks to the NIH's bold Blueprint for Neuroscience Research Initiative to launch several Cre-driver resource projects, as well as individual laboratories and private research organizations. With newly-discovered, genetically-encoded molecules that are capable of responding to sonar and magnetic stimulation, for sonogenetics or magnetogenetics, respectively, or detecting rapid voltage changes in neurons, Cre-lox neurogenetics will continue to aid brain research for years to come.

Functional analysis and molecular dynamics simulation of LOX-1 K167N polymorphism reveal alteration of receptor activity.

Directory of Open Access Journals (Sweden)

Silvia Biocca

Full Text Available The human lectin-like oxidized low density lipoprotein receptor 1 LOX-1, encoded by the ORL1 gene, is the major scavenger receptor for oxidized low density lipoprotein in endothelial cells. Here we report on the functional effects of a coding SNP, c.501G>C, which produces a single amino acid change (K>N at codon 167. Our study was aimed at elucidating whether the c.501G>C polymorphism changes the binding affinity of LOX-1 receptor altering its function. The presence of p.K167N mutation reduces ox-LDL binding and uptake. Ox-LDL activated extracellular signal-regulated kinases 1 and 2 (ERK 1/2 is inhibited. Furthermore, ox-LDL induced biosynthesis of LOX-1 receptors is dependent on the p.K167N variation. In human macrophages, derived from c.501G>C heterozygous individuals, the ox-LDL induced LOX-1 46 kDa band is markedly lower than in induced macrophages derived from c.501G>C controls. Investigation of p.K167N mutation through molecular dynamics simulation and electrostatic analysis suggests that the ox-LDL binding may be attributed to the coupling between the electrostatic potential distribution and the asymmetric flexibility of the basic spine residues. The N/N-LOX-1 mutant has either interrupted electrostatic potential and asymmetric fluctuations of the basic spine arginines.

Solid propellant ignition motors for LH_2/LOX rocket engine system

OpenAIRE

ARAKI, Tetsuo; AKIBA, Ryojiro; HASHIMOTO, Yasunari; AIHARA, Kenji; TOMITA, Etsu; YASUDA, Seiichi; 荒木, 哲夫; 秋葉, 鐐二郎; 橋本, 保成; 相原, 賢二; 富田, 悦; 安田, 誠一

1983-01-01

Solid propellant ignition motors are used in the series of experiments of the 10 ton LH_2/LOX engine featured by the channel wall thrust chamber, This paper presents design specification, experiments and results obtained by actual applications of those ignition motors.

Liquid oxygen LOX compatibility evaluations of aluminum lithium (Al-Li) alloys: Investigation of the Alcoa 2090 and MMC weldalite 049 alloys

Science.gov (United States)

Diwan, Ravinder M.

1989-01-01

The behavior of liquid oxygen (LOX) compatibility of aluminum lithium (Al-Li) alloys is investigated. Alloy systems of Alcoa 2090, vintages 1 to 3, and of Martin Marietta Corporation (MMC) Weldalite 049 were evaluated for their behavior related to the LOX compatibility employing liquid oxygen impact test conditions under ambient pressures and up to 1000 psi. The developments of these aluminum lithium alloys are of critical and significant interest because of their lower densities and higher specific strengths and improved mechanical properties at cryogenic temperatures. Of the different LOX impact tests carried out at the Marshall Space Flight Center (MSFC), it is seen that in certain test conditions at higher pressures, not all Al-Li alloys are LOX compatible. In case of any reactivity, it appears that lithium makes the material more sensitive at grain boundaries due to microstructural inhomogeneities and associated precipitate free zones (PFZ). The objectives were to identify and rationalize the microstructural mechanisms that could be relaxed to LOX compatibility behavior of the alloy system in consideration. The LOX compatibility behavior of Al-Li 2090 and Weldalite 049 is analyzed in detail using microstructural characterization techniques with light optical metallography, scanning electron microscopy (SEM), electron microprobe analysis, and surface studies using secondary ion mass spectrometry (SIMS), electron spectroscopy in chemical analysis (ESCA) and Auger electron spectroscopy (AES). Differences in the behavior of these aluminum lithium alloys are assessed and related to their chemistry, heat treatment conditions, and microstructural effects.

Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells.

Science.gov (United States)

Long, Qifang; Yang, Ru; Lu, Weixian; Zhu, Weipei; Zhou, Jundong; Zheng, Cui; Zhou, Dongmei; Yu, Ling; Wu, Jinchang

2017-01-01

Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells. The adenovirus (Ad)-CD133-Cre expressing Cre recombinase under the control of the CD133 promoter and Ad-CMV-LoxP-Neo-LoxP-tBid expressing tBid under the control of the CMV promoter were successfully constructed using the Cre/LoxP switching system. The co-infection of Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre selectively induced tBid overexpression, which inhibited cell growth and triggered the cell apoptosis of CD133+ ovarian cancer stem cells. The Cre/LoxP system-mediated tBid overexpression activated the pro-apoptotic signaling pathway and augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. Furthermore, in xenograft experiments, co-infection with the two recombinant adenoviruses markedly suppressed tumor growth in vivo and promoted cell apoptosis in tumor tissues. Taken together, the present study provides evidence that the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system can effectively eliminate CD133+ ovarian cancer stem cells, representing a novel therapeutic strategy for the treatment of ovarian cancer.

Influence of Lactobacillus plantarum WCFS1 on post-acidification, metabolite formation and survival of starter bacteria in set-yoghurt

NARCIS (Netherlands)

Settachaimongkon, Sarn; Valenberg, van Hein J.F.; Gazi, Inge; Nout, M.J.R.; Hooijdonk, van Toon C.M.; Zwietering, Marcel H.; Smid, Eddy J.

2016-01-01

The objectives of this study were to evaluate the growth and survival of the model probiotic strain Lactobacillus plantarum WCFS1 in co-culture with traditional yoghurt starters and to investigate the impact of preculturing on their survival and metabolite formation in set-yoghurt. L. plantarum

DNA adduct formation by the ubiquitous environmental pollutant 3-nitrobenzanthrone and its metabolites in rats

International Nuclear Information System (INIS)

Arlt, Volker M.; Sorg, Bernd L.; Osborne, Martin; Hewer, Alan; Seidel, Albrecht; Schmeiser, Heinz H.; Phillips, David H.

2003-01-01

Diesel exhaust is known to induce tumours in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust, 3-nitrobenzanthrone (3-NBA) is an extremely potent mutagen and suspected human carcinogen forming multiple DNA adducts in vitro. 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA), and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) were identified as 3-NBA metabolites. In order to gain insight into the pathways of metabolic activation leading to 3-NBA-derived DNA adducts we treated Wistar rats intraperitoneally with 2 mg/kg body weight of 3-NBA, 3-ABA, 3-Ac-ABA, or N-Ac-N-OH-ABA and compared DNA adducts present in different organs. With each compound either four or five DNA adduct spots were detected by TLC in all tissues examined (lung, liver, kidney, heart, pancreas, and colon) using the nuclease P1 or butanol enrichment version of the 32 P-postlabelling method, respectively. Using HPLC co-chromatographic analysis we showed that all major 3-NBA-DNA adducts produced in vivo in rats are derived from reductive metabolites bound to purine bases and lack an N-acetyl group. Our results indicate that 3-NBA metabolites (3-ABA, 3-Ac-ABA and N-Ac-N-OH-ABA) undergo several biotransformations and that N-hydroxy-3-aminobenzanthrone (N-OH-ABA) appears to be the common intermediate in 3-NBA-derived DNA adduct formation. Therefore, 3-NBA-DNA adducts are useful biomarkers for exposure to 3-NBA and its metabolites and may help to identify enzymes involved in their metabolic activation

Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae.

Science.gov (United States)

Choi, Ho-Jung; Kim, Yeon-Hee

2018-05-28

A Cre/ loxP -δ-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae . To allow repeated integrations, the reusable Candida glabrata MARKER ( CgMARKER ) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and β-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

Microsomal metabolism of trenbolone acetate metabolites: Transformation product formation and bioactivity.

Science.gov (United States)

Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbo...

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

Science.gov (United States)

Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2017-04-01

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

Science.gov (United States)

Rodriguez-Pascual, Fernando; Slatter, David Anthony

2016-11-23

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades. While LOX-generated collagen cross-linking metabolites have been detected in different metazoan families, there is limited information about when and how collagen acquired this particular modification. By analyzing telopeptide and helical sequences, we identified highly conserved, potential cross-linking sites throughout the metazoan tree of life. Based on this analysis, we propose that they have importantly contributed to the formation and further expansion of fibrillar collagens.

Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

International Nuclear Information System (INIS)

Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

2011-01-01

We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

OpenAIRE

Chu, Chih-Sheng; Wang, Yu-Chen; Lu, Long-Sheng; Walton, Brian; Yilmaz, H. Ramazan; Huang, Roger Y.; Sawamura, Tatsuya; Dixon, Richard A. F.; Lai, Wen-Ter; Chen, Chu-Huang; Lu, Jonathan

2013-01-01

Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL sa...

Arabidopsis CPR5 independently regulates seed germination and postgermination arrest of development through LOX pathway and ABA signaling.

Directory of Open Access Journals (Sweden)

Guilan Gao

Full Text Available The phytohormone abscisic acid (ABA and the lipoxygenases (LOXs pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently.

The mechanism of long non-coding RNA MEG3 for neurons apoptosis caused by hypoxia: mediated by miR-181b-12/15-LOX signaling pathway

Directory of Open Access Journals (Sweden)

Xiaomin Liu

2016-09-01

Full Text Available Objective: lncRNAs are recently thought to play a significant role in cellular homeostasis during pathological process of diseases by competing inhibiting miRNA function. The aim of present study was to assess the function of long non-coding RNA (lncRNA MEG3 and its functional interaction with microRNA-181b in cerebral ischemic infarct of mice and hypoxia-induced neurons apoptosis. Methods: To address this question, we performed the experiments with in vivo middle cerebral artery occlusion (MCAO mice model and in vitro oxygen-glucose deprivation (OGD-cultured neuronal HT22 cell line. Relative expression of MEG3, miR-181b and 12/15-LOX (lipoxygenase mRNA was determined using quantitative RT-PCR. Western blot was used to evaluate 12/15-LOX protein expression. TUNEL assay was performed to assess cell apoptosis.Results: In both MCAO mice and OGD-cultured HT22 cell, ischemia or hypoxia treatment results in a time-dependent increase in MEG3 and 12/15-LOX expression and decrease in miR-181b expression. Knockdown of MEG3 contributes to attenuation of hypoxia-induced apoptosis of HT22 cell. Also, expression level of MEG3 negatively correlated with miR-181b expression and positively correlated with 12/15-LOX expression. In contrary to MEG3, miR-181b overexpression attenuated hypoxia-induced HT22 cell apoptosis, as well as suppressed hypoxia-induced increase in 12/15-LOX expression. By luciferase reporter assay, we concluded that miR-181b directly binds to 12/15-LOX 3’-UTR, thereby negatively regulates 12/15-LOX expression. Conclusion: Our data suggested that long non-coding RNA MEG3 functions as a competing endogenous RNA for miR-181b to regulate 12/15-LOX expression in middle cerebral artery occlusion-induced ischemic infarct of brain nerve cells.

Metabolite profiling of microfluidic cell culture conditions for droplet based screening

DEFF Research Database (Denmark)

Björk, Sara M.; Sjoström, Staffan L.; Svahn, Helene Andersson

2015-01-01

We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets......, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast...... limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format....

LOX Gene Transcript Accumulation in Olive (Olea europaea L. Fruits at Different Stages of Maturation: Relationship between Volatile Compounds, Environmental Factors, and Technological Treatments for Oil Extraction

Directory of Open Access Journals (Sweden)

Innocenzo Muzzalupo

2012-01-01

Full Text Available The quality of olive oil is influenced by genetic and environmental factors and by the maturation state of drupes, but it is equally affected by technological treatments of the process. This work investigates the possible correlation between olive LOX gene transcript accumulation, evaluated in fruits collected at different stages of maturation, and chemical biomarkers of its activity. During olive fruit ripening, the same genotype harvested from two different farms shows a positive linear trend between LOX relative transcript accumulation and the content of volatile compounds present in the olive oil aroma. Interestingly, a negative linear trend was observed between LOX relative transcript accumulation and the content of volatile compounds present in the olive pastes obtained from olive fruits with and without malaxation. The changes in the olive LOX transcript accumulation reveal its environmental regulation and suggest differential physiological functions for the LOXs.

Rationalization and prediction of in vivo metabolite exposures: The role of metabolite kinetics, clearance predictions and in vitro parameters

Science.gov (United States)

Lutz, Justin D.; Fujioka, Yasushi; Isoherranen, Nina

2010-01-01

Importance of the field Due to growing concerns over toxic or active metabolites, significant efforts have been focused on qualitative identification of potential in vivo metabolites from in vitro data. However, limited tools are available to quantitatively predict their human exposures. Areas covered in this review Theory of clearance predictions and metabolite kinetics is reviewed together with supporting experimental data. In vitro and in vivo data of known circulating metabolites and their parent drugs was collected and the predictions of in vivo exposures of the metabolites were evaluated. What the reader will gain The theory and data reviewed will be useful in early identification of human metabolites that will circulate at significant levels in vivo and help in designing in vivo studies that focus on characterization of metabolites. It will also assist in rationalization of metabolite-to-parent ratios used as markers of specific enzyme activity. Take home message The relative importance of a metabolite in comparison to the parent compound as well as other metabolites in vivo can only be predicted using the metabolites in vitro formation and elimination clearances, and the in vivo disposition of a metabolite can only be rationalized when the elimination pathways of that metabolite are known. PMID:20557268

Selective methanol or formate production during continuous CO₂ fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool.

Science.gov (United States)

Tyurin, Michael; Kiriukhin, Michael

2013-09-01

Methanol-resistant mutant acetogen Clostridium sp. MT1424 originally producing only 365 mM acetate from CO₂/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach. Production of 2.2 M methanol was steady (p integrated cluster comprised only three copies of formate dehydrogenase the respective recombinants produced 95 mM formate (p < 0.005) under the same conditions. For commercialization, the suggested source of inorganic carbon would be CO₂ waste of IGCC power plant. Hydrogen may be produced in situ via powered by solar panels electrolysis.

Evaluation of 12-Lipoxygenase (12-LOX and Plasminogen Activator Inhibitor 1 (PAI-1 as Prognostic Markers in Prostate Cancer

Directory of Open Access Journals (Sweden)

Tomasz Gondek

2014-01-01

Full Text Available In carcinoma of prostate, a causative role of platelet 12-lipoxygenase (12-LOX and plasminogen activator inhibitor 1 (PAI-1 for tumor progression has been firmly established in tumor and/or adjacent tissue. Our goal was to investigate if 12-LOX and/or PAI-1 in patient’s plasma could be used to predict outcome of the disease. The study comprised 149 patients (age 70±9 divided into two groups: a study group with carcinoma confirmed by positive biopsy of prostate (n=116 and a reference group (n=33 with benign prostatic hyperplasia (BPH. The following parameters were determined by the laboratory test in plasma or platelet-rich plasma: protein level of 12-LOX, PAI-1, thromboglobulin (TGB, prostate specific antigen (PSA, C-reactive protein (CRP, hemoglobin (HGB, and hematocrit (HCT, as well as red (RBC and white blood cells (WBC, number of platelets (PLT, international normalized ratio of blood clotting (INR, and activated partial thromboplastin time (APTT. The only difference of significance was noticed in the concentration of 12-LOX in platelet rich plasma, which was lower in cancer than in BPH group. Standardization to TGB and platelet count increases the sensitivity of the test that might be used as a biomarker to assess risk for prostate cancer in periodically monitored patients.

Saturn V First Stage S-1C LOX Fuel Tanks

Science.gov (United States)

1960-01-01

This photograph shows the Saturn V assembled LOX (Liquid Oxygen) and fuel tanks ready for transport from the Manufacturing Engineering Laboratory at Marshall Space Flight Center in Huntsville, Alabama. The tanks were then shipped to the launch site at Kennedy Space Center for a flight. The towering 363-foot Saturn V was a multi-stage, multi-engine launch vehicle standing taller than the Statue of Liberty. Altogether, the Saturn V engines produced as much power as 85 Hoover Dams.

Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site.

Science.gov (United States)

Arguello, Tania; Moraes, Carlos T

2015-11-01

The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

CRISPR/Cas9-loxP-Mediated Gene Editing as a Novel Site-Specific Genetic Manipulation Tool.

Science.gov (United States)

Yang, Fayu; Liu, Changbao; Chen, Ding; Tu, Mengjun; Xie, Haihua; Sun, Huihui; Ge, Xianglian; Tang, Lianchao; Li, Jin; Zheng, Jiayong; Song, Zongming; Qu, Jia; Gu, Feng

2017-06-16

Cre-loxP, as one of the site-specific genetic manipulation tools, offers a method to study the spatial and temporal regulation of gene expression/inactivation in order to decipher gene function. CRISPR/Cas9-mediated targeted genome engineering technologies are sparking a new revolution in biological research. Whether the traditional site-specific genetic manipulation tool and CRISPR/Cas9 could be combined to create a novel genetic tool for highly specific gene editing is not clear. Here, we successfully generated a CRISPR/Cas9-loxP system to perform gene editing in human cells, providing the proof of principle that these two technologies can be used together for the first time. We also showed that distinct non-homologous end-joining (NHEJ) patterns from CRISPR/Cas9-mediated gene editing of the targeting sequence locates at the level of plasmids (episomal) and chromosomes. Specially, the CRISPR/Cas9-mediated NHEJ pattern in the nuclear genome favors deletions (64%-68% at the human AAVS1 locus versus 4%-28% plasmid DNA). CRISPR/Cas9-loxP, a novel site-specific genetic manipulation tool, offers a platform for the dissection of gene function and molecular insights into DNA-repair pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Utility of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) in the postmortem diagnosis of ischemic heart disease.

Science.gov (United States)

Takasu, Shojiro; Matsumoto, Sari; Kanto, Yuko; Iwadate, Kimiharu

2018-04-01

Ischemic heart disease (IHD) is a major cause of death in developed countries. Postmortem IHD diagnosis using biochemical markers is difficult because of the postmortem changes. In the present study, we investigated the utility of soluble lectin-like low-density lipoprotein receptor-1 (sLOX-1) in body fluids obtained from forensic autopsy cases. We measured pericardial fluid, urine, and serum sLOX-1 levels; these samples were obtained from medicolegal autopsy cases (n = 149, postmortem interval fluid and urine of patients with acute IHD had higher sLOX-1 levels (p fluid and urine samples obtained postmortem are useful markers of acute IHD. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Comparative hepatic in vitro depletion and metabolite formation of major perfluorooctane sulfonate precursors in Arctic polar bear, beluga whale, and ringed seal.

Science.gov (United States)

Letcher, Robert J; Chu, Shaogang; McKinney, Melissa A; Tomy, Gregg T; Sonne, Christian; Dietz, Rune

2014-10-01

Perfluorooctane sulfonate (PFOS) has been reported to be among the most concentrated persistent organic pollutants in Arctic marine wildlife. The present study examined the in vitro depletion of major PFOS precursors, N-ethyl-perfluorooctane sulfonamide (N-EtFOSA) and perfluorooctane sulfonamide (FOSA), as well as metabolite formation using an assay based on enzymatically viable liver microsomes for three top Arctic marine mammalian predators, polar bear (Ursus maritimus), beluga whale (Delphinapterus leucas), and ringed seal (Pusa hispida), and in laboratory rat (Rattus rattus) serving as a general mammalian model and positive control. Rat assays showed that N-EtFOSA (38 nM or 150 ng mL(-1)) to FOSA metabolism was >90% complete after 10 min, and at a rate of 23 pmol min(-1) mg(-1) protein. Examining all species in a full 90 min incubation assay, there was >95% N-EtFOSA depletion for the rat active control and polar bear microsomes, ∼65% for ringed seals, and negligible depletion of N-EtFOSA for beluga whale. Concomitantly, the corresponding in vitro formation of FOSA from N-EtFOSA was also quantitatively rat≈polar bear>ringed seal>beluga whale. A lack of enzymatic ability and/or a rate too slow to be detected likely explains the lack of N-EtFOSA to FOSA transformation for beluga whale. In the same assays, the depletion of the FOSA metabolite was insignificant (p>0.01) and with no concomitant formation of PFOS metabolite. This suggests that, in part, a source of FOSA is the biotransformation of accumulated N-EtFOSA in free-ranging Arctic ringed seal and polar bear. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Design and Activation of a LOX/GH Chemical Steam Generator

Science.gov (United States)

Saunders, G. P.; Mulkey, C. A.; Taylor, S. A.

2009-01-01

The purpose of this paper is to give a detailed description of the design and activation of the LOX/GH fueled chemical steam generator installed in Cell 2 of the E3 test facility at Stennis Space Center, MS (SSC). The steam generator uses a liquid oxygen oxidizer with gaseous hydrogen fuel. The combustion products are then quenched with water to create steam at pressures from 150 to 450 psig at temperatures from 350 to 750 deg F (from saturation to piping temperature limits).

LOX/hydrocarbon rocket engine analytical design methodology development and validation. Volume 2: Appendices

Science.gov (United States)

Niiya, Karen E.; Walker, Richard E.; Pieper, Jerry L.; Nguyen, Thong V.

1993-05-01

This final report includes a discussion of the work accomplished during the period from Dec. 1988 through Nov. 1991. The objective of the program was to assemble existing performance and combustion stability models into a usable design methodology capable of designing and analyzing high-performance and stable LOX/hydrocarbon booster engines. The methodology was then used to design a validation engine. The capabilities and validity of the methodology were demonstrated using this engine in an extensive hot fire test program. The engine used LOX/RP-1 propellants and was tested over a range of mixture ratios, chamber pressures, and acoustic damping device configurations. This volume contains time domain and frequency domain stability plots which indicate the pressure perturbation amplitudes and frequencies from approximately 30 tests of a 50K thrust rocket engine using LOX/RP-1 propellants over a range of chamber pressures from 240 to 1750 psia with mixture ratios of from 1.2 to 7.5. The data is from test configurations which used both bitune and monotune acoustic cavities and from tests with no acoustic cavities. The engine had a length of 14 inches and a contraction ratio of 2.0 using a 7.68 inch diameter injector. The data was taken from both stable and unstable tests. All combustion instabilities were spontaneous in the first tangential mode. Although stability bombs were used and generated overpressures of approximately 20 percent, no tests were driven unstable by the bombs. The stability instrumentation included six high-frequency Kistler transducers in the combustion chamber, a high-frequency Kistler transducer in each propellant manifold, and tri-axial accelerometers. Performance data is presented, both characteristic velocity efficiencies and energy release efficiencies, for those tests of sufficient duration to record steady state values.

Formation of reactive metabolites from benzene

International Nuclear Information System (INIS)

Snyder, R.; Jowa, L.; Witz, G.; Kalf, G.; Rushmore, T.

1986-01-01

Rat liver mitoplasts were incubated first with [ 3 H]dGTP, to form DNA labeled in G, and then with [ 14 C]benzene. The DNA was isolated and upon isopycnic density gradient centrifugation in CsCl yielded a single fraction of DNA labeled with both [ 3 H] and [ 14 C]. These data are consistent with the covalent binding of one or more metabolites of benzene to DNA. The DNA was enzymatically hydrolyzed to deoxynucleosides and chromatographed to reveal at least seven deoxyguanosine adducts. Further studies with labeled deoxyadenine revealed one adduct on deoxyadenine. [ 3 H]Deoxyguanosine was reacted with [ 14 C]hydroquinone or benzoquinone. The product was characterized using uv, fluorescence, mass and NMR spectroscopy. A proposed structure is described. (orig.)

Coil-On-Plug Ignition for LOX/Methane Liquid Rocket Engines in Thermal Vacuum Environments

Science.gov (United States)

Melcher, John C.; Atwell, Matthew J.; Morehead, Robert L.; Hurlbert, Eric A.; Bugarin, Luz; Chaidez, Mariana

2017-01-01

A coil-on-plug ignition system has been developed and tested for Liquid Oxygen (LOX) / liquid methane rocket engines operating in thermal vacuum conditions. The igniters were developed and tested as part of the Integrated Cryogenic Propulsion Test Article (ICPTA), previously tested as part of the Project Morpheus test vehicle. The ICPTA uses an integrated, pressure-fed, cryogenic LOX/methane propulsion system including a reaction control system (RCS) and a main engine. The ICPTA was tested at NASA Glenn Research Center's Plum Brook Station in the Spacecraft Propulsion Research Facility (B-2) under vacuum and thermal vacuum conditions. In order to successfully demonstrate ignition reliability in the vacuum conditions and eliminate corona discharge issues, a coil-on-plug ignition system has been developed. The ICPTA uses spark-plug ignition for both the main engine igniter and the RCS. The coil-on-plug configuration eliminates the conventional high-voltage spark plug cable by combining the coil and the spark-plug into a single component. Prior to ICPTA testing at Plum Brook, component-level reaction control engine (RCE) and main engine igniter testing was conducted at NASA Johnson Space Center (JSC), which demonstrated successful hot-fire ignition using the coil-on-plug from sea-level ambient conditions down to 10(exp.-2) torr. Integrated vehicle hot-fire testing at JSC demonstrated electrical and command/data system performance. Lastly, Plum Brook testing demonstrated successful ignitions at simulated altitude conditions at 30 torr and cold thermal-vacuum conditions at 6 torr. The test campaign successfully proved that coil-on-plug technology will enable integrated LOX/methane propulsion systems in future spacecraft.

Resistance to Fusarium verticillioides and fumonisin accumulation in maize inbred lines involves an earlier and enhanced expression of lipoxygenase (LOX) genes.

Science.gov (United States)

Maschietto, Valentina; Marocco, Adriano; Malachova, Alexandra; Lanubile, Alessandra

2015-09-01

Fusarium verticillioides causes ear rot in maize and contaminates the kernels with the fumonisin mycotoxins. It is known that plant lipoxygenase (LOX)-derived oxylipins regulate defence against pathogens and that the host-pathogen lipid cross-talk influences the pathogenesis. The expression profiles of fifteen genes of the LOX pathway were studied in kernels of resistant and susceptible maize lines, grown in field condition, at 3, 7 and 14 days post inoculation (dpi) with F. verticillioides. Plant defence responses were correlated with the pathogen growth, the expression profiles of fungal FUM genes for fumonisin biosynthesis and fumonisin content in the kernels. The resistant genotype limited fungal growth and fumonisin accumulation between 7 and 14 dpi. Pathogen growth became exponential in the susceptible line after 7 dpi, in correspondence with massive transcription of FUM genes and fumonisins augmented exponentially at 14 dpi. LOX pathway genes resulted strongly induced after pathogen inoculation in the resistant line at 3 and 7 dpi, whilst in the susceptible line the induction was reduced or delayed at 14 dpi. In addition, all genes resulted overexpressed before infection in kernels of the resistant genotype already at 3 dpi. The results suggest that resistance in maize may depend on an earlier activation of LOX genes and genes for jasmonic acid biosynthesis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Operational Issues in the Development of a Cost-Effective Reusable LOX/LH2 Engine

Science.gov (United States)

Ballard, Richard O.

2005-01-01

Contents include the following: SLI initiated under NASA Research Announcement (NRA) 8-30. Strategic Objectives. Make spaceflight safer (1 in 10000 mission LOV). Make spaceflight cheaper ($1000/lb payload). Two prototype LOX/LH2 engine systems funded under Cycle-1 of NRA8-30. COBRA (Pratt & Whitney / Aerojet). RS-83 (Rocketdyne).

Viral Cre-LoxP tools aid genome engineering in mammalian cells.

Science.gov (United States)

Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

2017-01-01

Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

Virtual Dual inhibition of COX-2 / 5-LOX enzymes based on binding properties of alpha-amyrins, the anti-inflammatory compound as a promising anti-cancer drug

Science.gov (United States)

Ranjbar, Mohammad Mehdi; Assadolahi, Vahideh; Yazdani, Mohsen; Nikaein, Donya; Rashidieh, Behnam

2016-01-01

Hydro-alcoholic fruit extract of Cordia myxa was considerably effective on curing acute inflammation in mouse model. Previous studies suggested significant anti-inflammatory activities as well as potential anticancer agent of α-amyrins in seeds. Inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipooxygenase (5-LOX) is significant in cancer prevention and therapeutics although this inhibition with chemo-drugs has its own side-effects. It is shown that these enzymes pathways are related to several cancers including colon, breast and lung cancer. This study was conducted based on Cordia species' α-amyrins as a safer natural anti-cancer compound for inhibition of COX-2 and 5-LOX enzymes by molecular docking. The X-ray crystal structure of COX2 / 5-LOX enzymes and α-amyrins was retrieved and energetically minimized respectively. The binding site and surface of enzymes were detected. Docking studies were performed by AutoDock 4.2 using Lamarckian genetic algorithm (LGA). Finally drug likeness, molecular pharmacokinetic properties and toxicity of α-amyrins was calculated. Molecular Docking revealed hydrogen and hydrophobic interactions between α-amyrins with both active sites of COX-2 and 5-LOX enzymes. Interestingly, it covalently bonded to Fe cofactor of 5-LOX enzyme and chelated this molecule. Base on binding energies (∆G) α-amyrin has more inhibitory effects on 5-LOX (-10.45 Kcal/mol) than COX-2 (-8.02 Kcal/mol). Analysis of molecular pharmacokinetic parameters suggested that α-amyrins complied with most sets of Lipinski's rules, and so it could be a suitable ligand for docking studies. Eventually, bioactivity score showed α-amyrins possess considerable biological activities as nuclear receptor, enzyme inhibitor, GPCR and protease inhibitor ligand. These results clearly demonstrate that α-amyrins could act as potential highly selective COX-/5-LOX inhibitor. Also, it is a safe compound in comparison with classical non-steroidal anti-inflammatory drugs (NSAIDs

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly

DEFF Research Database (Denmark)

Miller, Bryan W; Morton, Jennifer P; Pinese, Mark

2015-01-01

of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found...

Dietary exposure of juvenile common sole (Solea solea L.) to polybrominated diphenyl ethers (PBDEs): Part 2. Formation, bioaccumulation and elimination of hydroxylated metabolites

International Nuclear Information System (INIS)

Munschy, C.; Heas-Moisan, K.; Tixier, C.; Pacepavicius, G.; Alaee, M.

2010-01-01

The uptake, elimination and transformation of six PBDE congeners (BDE-28, -47, -99, -100, -153, -209) were studied in juvenile common sole (Solea solea L.) exposed to spiked contaminated food over a three-month period, and then depurated over a five-month period. Methoxylated (MeO-) and hydroxylated (OH-) PBDEs were determined in fish plasma exposed to PBDEs and compared to those obtained in control fish. While all MeO- and some OH- congeners identified in fish plasma were found to originate from non-metabolic sources, several OH- congeners, i.e., OH-tetraBDEs and OH-pentaBDEs, were found to originate from fish metabolism. Among these, 4'-OH-BDE-49 was identified as a BDE-47 metabolite. Congener 4'-OH-BDE-101, identified here for the first time, may be the result of BDE-99 metabolic transformation. Our results unequivocally showed that PBDEs are metabolised in juvenile sole via the formation of OH- metabolites. However, this was not a major biotransformation route compared to biotransformation through debromination. - Juvenile sole exposed to artificially-contaminated food showed the ability to biotransform PBDEs into hydroxylated metabolites; these meta-bolites accumulated in fish.

TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds.

Science.gov (United States)

Ma, Lei; Zhu, Fugui; Li, Zhenwei; Zhang, Jianfu; Li, Xin; Dong, Jiangli; Wang, Tao

2015-01-01

The deterioration of rice grain reduces the quality of rice, resulting in serious economic losses for farmers. Lipoxygenases (LOXs) catalyze the dioxygenation of polyunsaturated fatty acids with at least one cis,cis-1,4-pentadiene to form hydroperoxide, which is a major factor influencing seed longevity and viability. Recently, genome editing, an essential tool employed in reverse genetics, has been used experimentally to investigate basic plant biology or to modify crop plants for the improvement of important agricultural traits. In this study, we performed targeted mutagenesis in rice using transcription activator-like effector nucleases (TALENs) to improve seed storability. A modified ligation-independent cloning method (LIC) was employed to allow for the quick and efficient directional insertion of TALEN monomer modules into destination vectors used in plants. We demonstrated the feasibility and flexibility of the technology by developing a set of modular vectors for genome editing. After construction and validation, the TALEN pairs were used to create stable transgenic rice lines via Agrobacterium-mediated transformation. One heterozygous mutant (4%) was recovered from 25 transgenic NPTII-resistant lines, and the mutation was transmitted to the next generation. Further molecular and protein level experiments verified LOX3 deficiency and demonstrated the improvement of seed storability. Our work provides a flexible genome editing tool for improving important agronomic traits, as well as direct evidence that Lox3 has only a limited impact on seed longevity.

TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa Seeds.

Directory of Open Access Journals (Sweden)

Lei Ma

Full Text Available The deterioration of rice grain reduces the quality of rice, resulting in serious economic losses for farmers. Lipoxygenases (LOXs catalyze the dioxygenation of polyunsaturated fatty acids with at least one cis,cis-1,4-pentadiene to form hydroperoxide, which is a major factor influencing seed longevity and viability. Recently, genome editing, an essential tool employed in reverse genetics, has been used experimentally to investigate basic plant biology or to modify crop plants for the improvement of important agricultural traits. In this study, we performed targeted mutagenesis in rice using transcription activator-like effector nucleases (TALENs to improve seed storability. A modified ligation-independent cloning method (LIC was employed to allow for the quick and efficient directional insertion of TALEN monomer modules into destination vectors used in plants. We demonstrated the feasibility and flexibility of the technology by developing a set of modular vectors for genome editing. After construction and validation, the TALEN pairs were used to create stable transgenic rice lines via Agrobacterium-mediated transformation. One heterozygous mutant (4% was recovered from 25 transgenic NPTII-resistant lines, and the mutation was transmitted to the next generation. Further molecular and protein level experiments verified LOX3 deficiency and demonstrated the improvement of seed storability. Our work provides a flexible genome editing tool for improving important agronomic traits, as well as direct evidence that Lox3 has only a limited impact on seed longevity.

[Discover potential inhibitors of 5-LOX and LTA4H from Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix based on molecular simulation methods].

Science.gov (United States)

Gu, Yu; Zhang, Xu; Chen, Yan-Kun; Zhao, Bo-Wen; Zhang, Yan-Ling

2017-12-01

5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4H), as the major targets of 5-LOX branch in the arachidonic acid (AA) metabolic pathway, play an important role in the treatment of inflammation. Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix have clear anti-inflammation activities. In this paper, the targets of 5-LOX and LTA4H were used as the research carrier, and Hiphop module in DS4.0 (Discovery studio) was used to construct ingredients database for preliminary screening of three traditional Chinese medicines based on target inhibitor pharmacophore, so as to obtain 5-LOX and LTA4H potential active ingredients. The ingredients obtained in initial pharmacophore screening were further screened by using CDOCKER module, and the screening rules were established based on the score of initial compound and the key amino acids to obtain 12 potential 5-LOX inhibitors and 7 potential LTA4H inhibitors. To be more specific, the potential 5-LOX inhibitors included 6 ingredients in Rhei Radix et Rhizoma, such as procyanidins B2-3,3'-O-double gallate and revandchinone 2; four ingredients in notopterygium, such as dodecanoic acid and so on. On the other hand, potential LTA4H inhibitors included revandchinone 1, revandchinone 4 in Rhei Radix et Rhizoma, tridecanoic acid, tetracosanoic acid and methyl eicosanoate in Notopterygii Rhizoma et Radix, montanic acid methyl ester and N-docosanoyl-O-aminobenzoate in Genitana Macrophyllae Radix and so on. The molecular simulation methods were highly efficient and time-saving to obtain the potential inhibitors of 5-LOX and LTA4H, which could provide assistance for discovering the chemical quality indicators of anti-inflammatory efficacy of three Chinese herbs, and may be helpful to promote the whole-process quality control of three Chinese herbs. Copyright© by the Chinese Pharmaceutical Association.

Omega-3 polyunsaturated Fatty acids suppress the cystic lesion formation of peritoneal endometriosis in transgenic mouse models.

Directory of Open Access Journals (Sweden)

Kensuke Tomio

Full Text Available Omega-3 polyunsaturated fatty acids (omega-3 PUFAs play a role in controlling pathological inflammatory reactions. Endometriosis is characterized by the presence of endometrial tissue on the peritoneum and an exaggerated inflammatory environment around ectopic tissues. Here peritoneal endometriosis was reproduced using a mouse model in which murine endometrial fragments were inoculated into the peritoneal cavity of mice. Fat-1 mice, in which omega-6 can be converted to omega-3 PUFAs, or wild type mice, in which it cannot, were used for the endometriosis model to address the actions of omega-3 PUFAs on the development of endometriotic lesions. The number and weight of cystic endometriotic lesions in fat-1 mice two weeks after inoculation were significantly less than half to those of controls. Mediator lipidomics revealed that cystic endometriotic lesions and peritoneal fluids were abundant in 12/15-hydroxyeicosapentaenoic acid (12/15-HEPE, derived from eicosapentaenoic acid (EPA, and their amount in fat-1 mice was significantly larger than that in controls. 12/15-Lipoxygenase (12/15-LOX-knockout (KO and control mice with or without EPA administration were assessed for the endometriosis model. EPA administration decreased the number of lesions in controls but not in 12/15-LOX-KO mice. The peritoneal fluids in EPA-fed 12/15-LOX-KO mice contained reduced levels of EPA metabolites such as 12/15-HEPE and EPA-derived resolvin E3 even after EPA administration. cDNA microarrays of endometriotic lesions revealed that Interleukin-6 (IL-6 expression in fat-1 mice was significantly lower than that in controls. These results suggest that both endogenous and exogenous EPA-derived PUFAs protect against the development of endometriosis through their anti-inflammatory effects and, in particular, the 12/15-LOX-pathway products of EPA may be key mediators to suppress endometriosis.

Omega-3 polyunsaturated Fatty acids suppress the cystic lesion formation of peritoneal endometriosis in transgenic mouse models.

Science.gov (United States)

Tomio, Kensuke; Kawana, Kei; Taguchi, Ayumi; Isobe, Yosuke; Iwamoto, Ryo; Yamashita, Aki; Kojima, Satoko; Mori, Mayuyo; Nagamatsu, Takeshi; Arimoto, Takahide; Oda, Katsutoshi; Osuga, Yutaka; Taketani, Yuji; Kang, Jing X; Arai, Hiroyuki; Arita, Makoto; Kozuma, Shiro; Fujii, Tomoyuki

2013-01-01

Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) play a role in controlling pathological inflammatory reactions. Endometriosis is characterized by the presence of endometrial tissue on the peritoneum and an exaggerated inflammatory environment around ectopic tissues. Here peritoneal endometriosis was reproduced using a mouse model in which murine endometrial fragments were inoculated into the peritoneal cavity of mice. Fat-1 mice, in which omega-6 can be converted to omega-3 PUFAs, or wild type mice, in which it cannot, were used for the endometriosis model to address the actions of omega-3 PUFAs on the development of endometriotic lesions. The number and weight of cystic endometriotic lesions in fat-1 mice two weeks after inoculation were significantly less than half to those of controls. Mediator lipidomics revealed that cystic endometriotic lesions and peritoneal fluids were abundant in 12/15-hydroxyeicosapentaenoic acid (12/15-HEPE), derived from eicosapentaenoic acid (EPA), and their amount in fat-1 mice was significantly larger than that in controls. 12/15-Lipoxygenase (12/15-LOX)-knockout (KO) and control mice with or without EPA administration were assessed for the endometriosis model. EPA administration decreased the number of lesions in controls but not in 12/15-LOX-KO mice. The peritoneal fluids in EPA-fed 12/15-LOX-KO mice contained reduced levels of EPA metabolites such as 12/15-HEPE and EPA-derived resolvin E3 even after EPA administration. cDNA microarrays of endometriotic lesions revealed that Interleukin-6 (IL-6) expression in fat-1 mice was significantly lower than that in controls. These results suggest that both endogenous and exogenous EPA-derived PUFAs protect against the development of endometriosis through their anti-inflammatory effects and, in particular, the 12/15-LOX-pathway products of EPA may be key mediators to suppress endometriosis.

Classical Michaelis-Menten and system theory approach to modeling metabolite formation kinetics.

Science.gov (United States)

Popović, Jovan

2004-01-01

When single doses of drug are administered and kinetics are linear, techniques, which are based on the compartment approach and the linear system theory approach, in modeling the formation of the metabolite from the parent drug are proposed. Unlike the purpose-specific compartment approach, the methodical, conceptual and computational uniformity in modeling various linear biomedical systems is the dominant characteristic of the linear system approach technology. Saturation of the metabolic reaction results in nonlinear kinetics according to the Michaelis-Menten equation. The two compartment open model with Michaelis-Menten elimination kinetics is theorethicaly basic when single doses of drug are administered. To simulate data or to fit real data using this model, one must resort to numerical integration. A biomathematical model for multiple dosage regimen calculations of nonlinear metabolic systems in steady-state and a working example with phenytoin are presented. High correlation between phenytoin steady-state serum levels calculated from individual Km and Vmax values in the 15 adult epileptic outpatients and the observed levels at the third adjustment of phenytoin daily dose (r=0.961, p<0.01) were found.

Laser ignition of a multi-injector LOX/methane combustor

Science.gov (United States)

Börner, Michael; Manfletti, Chiara; Hardi, Justin; Suslov, Dmitry; Kroupa, Gerhard; Oschwald, Michael

2018-06-01

This paper reports the results of a test campaign of a laser-ignited combustion chamber with 15 shear coaxial injectors for the propellant combination LOX/methane. 259 ignition tests were performed for sea-level conditions. The igniter based on a monolithic ceramic laser system was directly attached to the combustion chamber and delivered 20 pulses with individual pulse energies of {33.2 ± 0.8 mJ } at 1064 nm wavelength and 2.3 ns FWHM pulse length. The applicability, reliability, and reusability of this ignition technology are demonstrated and the associated challenges during the start-up process induced by the oxygen two-phase flow are formulated. The ignition quality and pressure dynamics are evaluated using 14 dynamic pressure sensors distributed both azimuthally and axially along the combustion chamber wall. The influence of test sequencing on the ignition process is briefly discussed and the relevance of the injection timing of the propellants for the ignition process is described. The flame anchoring and stabilization process, as monitored using an optical probe system close to the injector faceplate connected to photomultiplier elements, is presented. For some of the ignition tests, non-uniform anchoring was detected with no influence onto the anchoring at steady-state conditions. The non-uniform anchoring can be explained by the inhomogeneous, transient injection of the two-phase flow of oxygen across the faceplate. This characteristic is verified by liquid nitrogen cold flow tests that were recorded by high-speed imaging. We conclude that by adapting the ignition sequence, laser ignition by optical breakdown of the propellants within the shear layer of a coaxial shear injector is a reliable ignition technology for LOX/methane combustors without significant over-pressure levels.

Laser ignition of a multi-injector LOX/methane combustor

Science.gov (United States)

Börner, Michael; Manfletti, Chiara; Hardi, Justin; Suslov, Dmitry; Kroupa, Gerhard; Oschwald, Michael

2018-02-01

This paper reports the results of a test campaign of a laser-ignited combustion chamber with 15 shear coaxial injectors for the propellant combination LOX/methane. 259 ignition tests were performed for sea-level conditions. The igniter based on a monolithic ceramic laser system was directly attached to the combustion chamber and delivered 20 pulses with individual pulse energies of {33.2 ± 0.8 mJ } at 1064 nm wavelength and 2.3 ns FWHM pulse length. The applicability, reliability, and reusability of this ignition technology are demonstrated and the associated challenges during the start-up process induced by the oxygen two-phase flow are formulated. The ignition quality and pressure dynamics are evaluated using 14 dynamic pressure sensors distributed both azimuthally and axially along the combustion chamber wall. The influence of test sequencing on the ignition process is briefly discussed and the relevance of the injection timing of the propellants for the ignition process is described. The flame anchoring and stabilization process, as monitored using an optical probe system close to the injector faceplate connected to photomultiplier elements, is presented. For some of the ignition tests, non-uniform anchoring was detected with no influence onto the anchoring at steady-state conditions. The non-uniform anchoring can be explained by the inhomogeneous, transient injection of the two-phase flow of oxygen across the faceplate. This characteristic is verified by liquid nitrogen cold flow tests that were recorded by high-speed imaging. We conclude that by adapting the ignition sequence, laser ignition by optical breakdown of the propellants within the shear layer of a coaxial shear injector is a reliable ignition technology for LOX/methane combustors without significant over-pressure levels.

Influence of Lactobacillus plantarum WCFS1 on post-acidification, metabolite formation and survival of starter bacteria in set-yoghurt.

Science.gov (United States)

Settachaimongkon, Sarn; van Valenberg, Hein J F; Gazi, Inge; Nout, M J Robert; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J

2016-10-01

The objectives of this study were to evaluate the growth and survival of the model probiotic strain Lactobacillus plantarum WCFS1 in co-culture with traditional yoghurt starters and to investigate the impact of preculturing on their survival and metabolite formation in set-yoghurt. L. plantarum WCFS1 was precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor before inoculation in milk. Adaptive responses of L. plantarum WCFS1 were evaluated by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated that sublethal preculturing did not significantly affect survival of L. plantarum WCFS1. On the other hand, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of Lactobacillus delbrueckii subsp. bulgaricus which consequently reduced the post-acidification of yoghurt during refrigerated storage. A complementary metabolomics approach using headspace SPME-GC/MS and (1)H NMR combined with multivariate statistical analysis revealed substantial impact of sublethally precultured L. plantarum WCFS1 on the metabolite profiles of set-yoghurt. This study provides insight in the technological implications of non-dairy model probiotic strain L. plantarum WCFS1, such as its good stability in fermented milk and the inhibitory effect on post-acidification. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

Science.gov (United States)

Chakraborti, Dipankar; Sarkar, Anindya; Mondal, Hossain A; Schuermann, David; Hohn, Barbara; Sarmah, Bidyut K; Das, Sampa

2008-10-01

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

Neonatal Maturation of Paracetamol (Acetaminophen) Glucuronidation, Sulfation, and Oxidation Based on a Parent-Metabolite Population Pharmacokinetic Model.

Science.gov (United States)

Cook, Sarah F; Stockmann, Chris; Samiee-Zafarghandy, Samira; King, Amber D; Deutsch, Nina; Williams, Elaine F; Wilkins, Diana G; Sherwin, Catherine M T; van den Anker, John N

2016-11-01

This study aimed to model the population pharmacokinetics of intravenous paracetamol and its major metabolites in neonates and to identify influential patient characteristics, especially those affecting the formation clearance (CL formation ) of oxidative pathway metabolites. Neonates with a clinical indication for intravenous analgesia received five 15-mg/kg doses of paracetamol at 12-h intervals (paracetamol, paracetamol-glucuronide, paracetamol-sulfate, and the combined oxidative pathway metabolites (paracetamol-cysteine and paracetamol-N-acetylcysteine) were simultaneously modeled in NONMEM 7.2. The model incorporated 259 plasma and 350 urine samples from 35 neonates with a mean gestational age of 33.6 weeks (standard deviation 6.6). CL formation for all metabolites increased with weight; CL formation for glucuronidation and oxidation also increased with postnatal age. At the mean weight (2.3 kg) and postnatal age (7.5 days), CL formation estimates (bootstrap 95% confidence interval; between-subject variability) were 0.049 L/h (0.038-0.062; 62 %) for glucuronidation, 0.21 L/h (0.17-0.24; 33 %) for sulfation, and 0.058 L/h (0.044-0.078; 72 %) for oxidation. Expression of individual oxidation CL formation as a fraction of total individual paracetamol clearance showed that, on average, fractional oxidation CL formation increased paracetamol and its metabolites in neonates. Maturational changes in the fraction of paracetamol undergoing oxidation were small relative to between-subject variability.

12/15 Lipoxygenase regulation of colorectal tumorigenesis is determined by the relative tumor levels of its metabolite 12-HETE and 13-HODE in animal models.

Science.gov (United States)

Chang, Jian; Jiang, Li; Wang, Yinqiu; Yao, Bing; Yang, Shilin; Zhang, Bixiang; Zhang, Ming-Zhi

2015-02-20

Colorectal cancer (CRC) continues to be a major cause of morbidity and mortality. The arachidonic acid (AA) pathway and linoleic acid (LA) pathway have been implicated as important contributors to CRC development and growth. Human 15-lipoxygenase 1 (15-LOX-1) converts LA to anti-tumor 13-S-hydroxyoctadecadienoic acid (13-HODE)and 15-LOX-2 converts AA to 15-hydroxyeicosatetraenoic acid (15-HETE). In addition, human 12-LOX metabolizes AA to pro-tumor 12-HETE. In rodents, the function of 12-LOX and 15-LOX-1 and 15-LOX-2 is carried out by a single enzyme, 12/15-LOX. As a result, conflicting conclusions concerning the role of 12-LOX and 15-LOX have been obtained in animal studies. In the present studies, we determined that PD146176, a selective 15-LOX-1 inhibitor, markedly suppressed 13-HODE generation in human colon cancer HCA-7 cells and HCA-7 tumors, in association with increased tumor growth. In contrast, PD146176 treatment led to decreases in 12-HETE generation in mouse colon cancer MC38 cells and MC38 tumors, in association with tumor inhibition. Surprisingly, deletion of host 12/15-LOX alone led to increased MC38 tumor growth, in association with decreased tumor 13-HODE levels, possibly due to inhibition of 12/15-LOX activity in stroma. Therefore, the effect of 12/15-LOX on colorectal tumorigenesis in mouse models could be affected by tumor cell type (human or mouse), relative 12/15 LOX activity in tumor cells and stroma as well as the relative tumor 13-HODE and 12-HETE levels.

Biotransformation of Daclatasvir In Vitro and in Nonclinical Species: Formation of the Main Metabolite by Pyrrolidine δ-Oxidation and Rearrangement.

Science.gov (United States)

Li, Wenying; Zhao, Weiping; Liu, Xiaohong; Huang, Xiaohua; Lopez, Omar D; Leet, John E; Fancher, R Marcus; Nguyen, Van; Goodrich, Jason; Easter, John; Hong, Yang; Caceres-Cortes, Janet; Chang, Shu Y; Ma, Li; Belema, Makonen; Hamann, Lawrence G; Gao, Min; Zhu, Mingshe; Shu, Yue-Zhong; Humphreys, W Griffith; Johnson, Benjamin M

2016-06-01

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

Science.gov (United States)

Lennard, Lynne; Cartwright, Cher Suzanne; Wade, Rachel; Richards, Susan M; Vora, Ajay

2013-01-01

Aims In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype–genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. Methods We measured TPMT (activity, as units ml−1 packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 108 RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Results Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. Conclusions In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy. PMID:23252716

Prediction of Relative In Vivo Metabolite Exposure from In Vitro Data Using Two Model Drugs: Dextromethorphan and Omeprazole

Science.gov (United States)

Lutz, Justin D.

2012-01-01

Metabolites can have pharmacological or toxicological effects, inhibit metabolic enzymes, and be used as probes of drug-drug interactions or specific cytochrome P450 (P450) phenotypes. Thus, better understanding and prediction methods are needed to characterize metabolite exposures in vivo. This study aimed to test whether in vitro data could be used to predict and rationalize in vivo metabolite exposures using two model drugs and P450 probes: dextromethorphan and omeprazole with their primary metabolites dextrorphan, 5-hydroxyomeprazole (5OH-omeprazole), and omeprazole sulfone. Relative metabolite exposures were predicted using metabolite formation and elimination clearances. For dextrorphan, the formation clearances of dextrorphan glucuronide and 3-hydroxymorphinan from dextrorphan in human liver microsomes were used to predict metabolite (dextrorphan) clearance. For 5OH-omeprazole and omeprazole sulfone, the depletion rates of the metabolites in human hepatocytes were used to predict metabolite clearance. Dextrorphan/dextromethorphan in vivo metabolite/parent area under the plasma concentration versus time curve ratio (AUCm/AUCp) was overpredicted by 2.1-fold, whereas 5OH-omeprazole/omeprazole and omeprazole sulfone/omeprazole were predicted within 0.75- and 1.1-fold, respectively. The effect of inhibition or induction of the metabolite's formation and elimination on the AUCm/AUCp ratio was simulated. The simulations showed that unless metabolite clearance pathways are characterized, interpretation of the metabolic ratios is exceedingly difficult. This study shows that relative in vivo metabolite exposure can be predicted from in vitro data and characterization of secondary metabolism of probe metabolites is critical for interpretation of phenotypic data. PMID:22010218

Three-Dimensional Numerical Analysis of LOX/Kerosene Engine Exhaust Plume Flow Field Characteristics

Directory of Open Access Journals (Sweden)

Hong-hua Cai

2017-01-01

Full Text Available Aiming at calculating and studying the flow field characteristics of engine exhaust plume and comparative analyzing the effects of different chemical reaction mechanisms on the engine exhaust plume flow field characteristics, a method considering fully the combustion state influence is put forward, which is applied to exhaust plume flow field calculation of multinozzle engine. On this basis, a three-dimensional numerical analysis of the effects of different chemical reaction mechanisms on LOX/kerosene engine exhaust plume flow field characteristics was carried out. It is found that multistep chemical reaction can accurately describe the combustion process in the LOX/kerosene engine, the average chamber pressure from the calculation is 4.63% greater than that of the test, and the average chamber temperature from the calculation is 3.34% greater than that from the thermodynamic calculation. The exhaust plumes of single nozzle and double nozzle calculated using the global chemical reaction are longer than those using the multistep chemical reaction; the highest temperature and the highest velocity on the plume axis calculated using the former are greater than that using the latter. The important influence of chemical reaction mechanism must be considered in the study of the fixing structure of double nozzle engine on the rocket body.

Celecoxib use and circulating oxylipins in a colon polyp prevention trial.

Directory of Open Access Journals (Sweden)

Jessica A Martinez

Full Text Available Drugs that inhibit cyclooxygenase (COX-2 and the metabolism of arachidonic acid (ARA to prostaglandin E2 are potent anti-inflammatory agents used widely in the treatment of joint and muscle pain. Despite their benefits, daily use of these drugs has been associated with hypertension, cardiovascular and gastrointestinal toxicities. It is now recognized that ARA is metabolized to a number of bioactive oxygenated lipids (oxylipins by cyclooxygenase (COX, lipoxygenase (LOX, and cytochrome P450 (CYP450 enzymes. Currently, the contribution of individual variability in ARA metabolism in response to the COX-2 inhibitors and potential adverse effects remains poorly understood. Using patient samples from the randomized, placebo-controlled phase III selenium/celecoxib (Sel/Cel trial for the prevention of colorectal adenomatous polyps, we analyzed plasma concentrations of 74 oxylipins in a subset of participants who received celecoxib (n = 90 or placebo (n = 95. We assessed the effect of celecoxib (with and without low dose aspirin on circulating oxylipins and systolic blood pressure (SBP. Individual CYP450- and LOX- but not COX-derived metabolites were higher with celecoxib than placebo (P<0.05 and differences were greater among non-aspirin users. LOX derived 5- and 8-HETE were elevated with celecoxib and positively associated with systolic blood pressure (P = 0.011 and P = 0.019 respectively. 20-HETE, a prohypertensive androgen-sensitive CYP450 metabolite was higher with celecoxib absent aspirin and was positively associated with SBP in men (P = 0.040 but not women. Independent of celecoxib or aspirin, LOX derived metabolites from ARA were strongly associated with SBP including 5- and 8-HETE. These findings support oxylipins, particularly the ARA LOX-derived, in blood pressure control and indicate that pharmacologic inhibition of COX-2 has effects on LOX and CYP450 ARA metabolism that contribute to hypertension in some patients.

Regional gene expression of LOX-1, VCAM-1, and ICAM-1 in aorta of HIV-1 transgenic rats

DEFF Research Database (Denmark)

Hag, Anne Mette Fisker; Kristoffersen, Ulrik Sloth; Pedersen, Sune Folke

2009-01-01

endpoints, studies in animal models could be attractive alternatives. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in HIV-1...... transgenic (HIV-1Tg) rats; these genes are all thought to play important roles in early atherogenesis. Furthermore, the plasma level of sICAM-1 was measured. We found that gene expressions of LOX-1 and VCAM-1 were higher in the aortic arch of HIV-1Tg rats compared to controls. Also, the level of sICAM-1......-infection per se may cause atherosclerosis. This transgenic rat model may be a very promising model for further studies of the pathophysiology behind HIV-associated cardiovascular disease....

LOX-1 and Its Splice Variants: A New Challenge for Atherosclerosis and Cancer-Targeted Therapies

Science.gov (United States)

Rizzacasa, Barbara; Morini, Elena; Pucci, Sabina; Murdocca, Michela; Novelli, Giuseppe; Amati, Francesca

2017-01-01

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both “risk” and “protective” splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target. PMID:28146073

Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites

Energy Technology Data Exchange (ETDEWEB)

Palumbo, Fabrizio [Instituto de Tecnología Química UPV-CSIC/Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia (Spain); Garcia-Lainez, Guillermo [Instituto de Investigación Sanitaria (IIS) La Fe, Hospital Universitari i Politècnic La Fe, Avenida de Fernando Abril Martorell 106, 46026 Valencia (Spain); Limones-Herrero, Daniel [Instituto de Tecnología Química UPV-CSIC/Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia (Spain); Coloma, M. Dolores; Escobar, Javier [Instituto de Investigación Sanitaria (IIS) La Fe, Hospital Universitari i Politècnic La Fe, Avenida de Fernando Abril Martorell 106, 46026 Valencia (Spain); Jiménez, M. Consuelo [Instituto de Tecnología Química UPV-CSIC/Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia (Spain); Miranda, Miguel A., E-mail: mmiranda@qim.upv.es [Instituto de Tecnología Química UPV-CSIC/Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia (Spain); and others

2016-12-15

Chlorpromazine (CPZ) is an anti-psychotic drug widely used to treat disorders such as schizophrenia or manic-depression. Unfortunately, CPZ exhibits undesirable side effects such as phototoxic and photoallergic reactions in humans. In general, the influence of drug metabolism on this type of reactions has not been previously considered in photosafety testing. Thus, the present work aims to investigate the possible photo(geno)toxic potential of drug metabolites, using CPZ as an established reference compound. In this case, the metabolites selected for the study are demethylchlorpromazine (DMCPZ), didemethylchlorpromazine (DDMCPZ) and chlorpromazine sulfoxide (CPZSO). The demethylated CPZ metabolites DMCPZ and DDMCPZ maintain identical chromophore to the parent drug. In this work, it has been found that the nature of the aminoalkyl side chain modulates the hydrophobicity and the photochemical properties (for instance, the excited state lifetimes), but it does not change the photoreactivity pattern, which is characterized by reductive photodehalogenation, triggered by homolytic carbon-chlorine bond cleavage with formation of highly reactive aryl radical intermediates. Accordingly, these metabolites are phototoxic to cells, as revealed by the 3T3 NRU assay; their photo-irritation factors are even higher than that of CPZ. The same trend is observed in photogenotoxicity studies, both with isolated and with cellular DNA, where DMCPZ and DDMCPZ are more active than CPZ itself. In summary, side-chain demethylation of CPZ, as a consequence of Phase I biotransformation, does not result a photodetoxification. Instead, it leads to metabolites that exhibit in an even enhanced photo(geno)toxicity. - Highlights: • Demethylated CPZ metabolites are phototoxic to cells, as revealed by the NRU assay. • Single cell electrophoresis (Comet Assay) confirms the photodamage to cellular DNA. • DNA single strand breaks formation is observed on agarose gel electrophoresis. �

Enhanced photo(geno)toxicity of demethylated chlorpromazine metabolites

International Nuclear Information System (INIS)

Palumbo, Fabrizio; Garcia-Lainez, Guillermo; Limones-Herrero, Daniel; Coloma, M. Dolores; Escobar, Javier; Jiménez, M. Consuelo; Miranda, Miguel A.

2016-01-01

Chlorpromazine (CPZ) is an anti-psychotic drug widely used to treat disorders such as schizophrenia or manic-depression. Unfortunately, CPZ exhibits undesirable side effects such as phototoxic and photoallergic reactions in humans. In general, the influence of drug metabolism on this type of reactions has not been previously considered in photosafety testing. Thus, the present work aims to investigate the possible photo(geno)toxic potential of drug metabolites, using CPZ as an established reference compound. In this case, the metabolites selected for the study are demethylchlorpromazine (DMCPZ), didemethylchlorpromazine (DDMCPZ) and chlorpromazine sulfoxide (CPZSO). The demethylated CPZ metabolites DMCPZ and DDMCPZ maintain identical chromophore to the parent drug. In this work, it has been found that the nature of the aminoalkyl side chain modulates the hydrophobicity and the photochemical properties (for instance, the excited state lifetimes), but it does not change the photoreactivity pattern, which is characterized by reductive photodehalogenation, triggered by homolytic carbon-chlorine bond cleavage with formation of highly reactive aryl radical intermediates. Accordingly, these metabolites are phototoxic to cells, as revealed by the 3T3 NRU assay; their photo-irritation factors are even higher than that of CPZ. The same trend is observed in photogenotoxicity studies, both with isolated and with cellular DNA, where DMCPZ and DDMCPZ are more active than CPZ itself. In summary, side-chain demethylation of CPZ, as a consequence of Phase I biotransformation, does not result a photodetoxification. Instead, it leads to metabolites that exhibit in an even enhanced photo(geno)toxicity. - Highlights: • Demethylated CPZ metabolites are phototoxic to cells, as revealed by the NRU assay. • Single cell electrophoresis (Comet Assay) confirms the photodamage to cellular DNA. • DNA single strand breaks formation is observed on agarose gel electrophoresis. �

A revolutionary lunar space transportation system architecture using extraterrestrial LOX-augmented NTR propulsion

Science.gov (United States)

Borowski, Stanley K.; Corban, Robert R.; Culver, Donald W.; Bulman, Melvin J.; McIlwain, Mel C.

1994-08-01

The concept of a liquid oxygen (LOX)-augmented nuclear thermal rocket (NTR) engine is introduced, and its potential for revolutionizing lunar space transportation system (LTS) performance using extraterrestrial 'lunar-derived' liquid oxygen (LUNOX) is outlined. The LOX-augmented NTR (LANTR) represents the marriage of conventional liquid hydrogen (LH2)-cooled NTR and airbreathing engine technologies. The large divergent section of the NTR nozzle functions as an 'afterburner' into which oxygen is injected and supersonically combusted with nuclear preheated hydrogen emerging from the NTR's choked sonic throat: 'scramjet propulsion in reverse.' By varying the oxygen-to-fuel mixture ratio (MR), the LANTR concept can provide variable thrust and specific impulse (Isp) capability with a LH2-cooled NTR operating at relatively constant power output. For example, at a MR = 3, the thrust per engine can be increased by a factor of 2.75 while the Isp decreases by only 30 percent. With this thrust augmentation option, smaller, 'easier to develop' NTR's become more acceptable from a mission performance standpoint (e.g., earth escape gravity losses are reduced and perigee propulsion requirements are eliminated). Hydrogen mass and volume is also reduced resulting in smaller space vehicles. An evolutionary NTR-based lunar architecture requiring only Shuttle C and/or 'in-line' shuttle-derived launch vehicles (SDV's) would operate initially in an 'expandable mode' with NTR lunar transfer vehicles (LTV's) delivering 80 percent more payload on piloted missions than their LOX/LH2 chemical propulsion counterparts. With the establishment of LUNOX production facilities on the lunar surface and 'fuel/oxidizer' depot in low lunar orbit (LLO), monopropellant NTR's would be outfitted with an oxygen propellant module, feed system, and afterburner nozzle for 'bipropellant' operation. The LANTR cislunar LTV now transitions to a reusable mode with smaller vehicle and payload doubling benefits on

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems.

Science.gov (United States)

Liu, Ju; Deutsch, Urban; Fung, Iris; Lobe, Corrinne G

2014-11-01

In the present study, the tetracycline-off and Cre/ loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP -flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

Directory of Open Access Journals (Sweden)

VanDoorn Arjen

2010-08-01

Full Text Available Abstract Background Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants. Results The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu, was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3 and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene in irlox2 plants. Conclusions The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential

Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

OpenAIRE

Cox, Brandon C.; Liu, Zhiyong; Lagarde, Marcia M. Mellado; Zuo, Jian

2012-01-01

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreERTM and Atoh1-CreERT2 a...

Metabolites in vertebrate Hedgehog signaling.

Science.gov (United States)

Roberg-Larsen, Hanne; Strand, Martin Frank; Krauss, Stefan; Wilson, Steven Ray

2014-04-11

The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibition of 5-LOX, COX-1, and COX-2 increases tendon healing and reduces muscle fibrosis and lipid accumulation after rotator cuff repair.

Science.gov (United States)

Oak, Nikhil R; Gumucio, Jonathan P; Flood, Michael D; Saripalli, Anjali L; Davis, Max E; Harning, Julie A; Lynch, Evan B; Roche, Stuart M; Bedi, Asheesh; Mendias, Christopher L

2014-12-01

The repair and restoration of function after chronic rotator cuff tears are often complicated by muscle atrophy, fibrosis, and fatty degeneration of the diseased muscle. The inflammatory response has been implicated in the development of fatty degeneration after cuff injuries. Licofelone is a novel anti-inflammatory drug that inhibits 5-lipoxygenase (5-LOX), as well as cyclooxygenase (COX)-1 and COX-2 enzymes, which play important roles in inducing inflammation after injuries. While previous studies have demonstrated that nonsteroidal anti-inflammatory drugs and selective inhibitors of COX-2 (coxibs) may prevent the proper healing of muscles and tendons, studies about bone and cartilage have demonstrated that drugs that inhibit 5-LOX concurrently with COX-1 and COX-2 may enhance tissue regeneration. After the repair of a chronic rotator cuff tear in rats, licofelone would increase the load to failure of repaired tendons and increase the force production of muscle fibers. Controlled laboratory study. Rats underwent supraspinatus release followed by repair 28 days later. After repair, rats began a treatment regimen of either licofelone or a vehicle for 14 days, at which time animals were euthanized. Supraspinatus muscles and tendons were then subjected to contractile, mechanical, histological, and biochemical analyses. Compared with controls, licofelone-treated rats had a grossly apparent decrease in inflammation and increased fibrocartilage formation at the enthesis, along with a 62% increase in the maximum load to failure and a 51% increase in peak stress to failure. Licofelone resulted in a marked reduction in fibrosis and lipid content in supraspinatus muscles as well as reduced expression of several genes involved in fatty infiltration. Despite the decline in fibrosis and fat accumulation, muscle fiber specific force production was reduced by 23%. The postoperative treatment of cuff repair with licofelone may reduce fatty degeneration and enhance the development

Efecto in vitro del D-002 sobre la actividad enzimática de la 5-lipoxigenasa (5-LOX

Directory of Open Access Journals (Sweden)

Yohani Pérez Guerra

2012-06-01

Full Text Available Introducción: el D-002, mezcla de seis alcoholes alifáticos primarios de alto peso molecular purificada de la cera de abejas, ha mostrado efectos antiinflamatorios, sin gastrotoxicidad secundaria, enmodelos experimentales. El tratamiento oral con D-002 reduce las concentraciones de leucotrieno B4 (LTB4 en exudados pleurales de ratas con pleuresía por carragenina, sugiriendo que podría inhibir la actividad enzimática de la 5-lipooxigenasa (5-LOX, si bien los mecanismos de la acción antiinflamatoria del D-002 no habían sido aún explorados. Objetivo: evaluar el efecto in vitro del D-002 sobre la actividad de la 5-LOX, utilizando la fracción citosólica de homogenatos de hígado de ratas. Métodos: las condiciones de ensayo utilizadas fueron las siguientes: fracción citosólica (50 µg de proteína disuelta en solución reguladora borato 0,2 mol/L (pH 9 y ácido linoleico (7,8-250 mmol/L como sustrato, ensayándose muestras paralelas incubadas con Tween-20/H2O (2 % (vehículo, muestras controles, D-002 (0,9-1 000 µg/mL o Lyprinol (500 µg/mL (sustancia de referencia. La actividad enzimática se evaluó mediante el cambio de absorbancia a 234 nm producido por la formación de dienos conjugados y medido en espectrofotómetro UV-visible. Resultados: la adición in vitro del D-002 produjo una inhibición significativa, dependiente de la dosis (r= 0,980; p< 0,001 (IC50= 95,34 µg/mL e incompetitiva de la actividad de la 5-LOX, cuya inhibición máxima (70 % se alcanzó con 500 µg/mL. Conclusiones: con este estudio se demuestra que el D-002 es capaz de inhibir la actividad enzimática de la 5-LOX, efecto que podría explicar, al menos parcialmente, su acción antiinflamatoria en modelos experimentales in vivo.

Efecto in vitro del D-003 sobre la actividad enzimática de la 5-lipoxigenasa (5-LOX

Directory of Open Access Journals (Sweden)

Yohani Pérez Guerra

Full Text Available Introducción: el D-003, mezcla de ácidos alifáticos primarios superiores purificada de la cera de la caña, inhibe la síntesis de colesterol. Trabajos recientes han demostrado que el D-003 resulta efectivo en modelos experimentales de osteoartritis y que inhibe la actividad de las enzimas COX1 y COX2, preferentemente la COX1, sin producir gastrotoxicidad. Ha sido referido que los inhibidores duales de las enzimas COX y 5-LOX presentan efectos antinflamatorios desprovistos de gastrotoxicidad o que incluso, pueden resultar gastroprotectores. De acuerdo con estos antecedentes, el D-003 podría ser un inhibidor dual de dichas enzimas. Objetivo: investigar el efecto in vitro del D-003 sobre la actividad enzimática de la 5-LOX, utilizando la fracción citosólica de leucocitos polimorfonucleares de ratas. Métodos: se utilizaron las condiciones de ensayo siguientes: fracción citosólica (50 µg de proteína disuelta en solución reguladora borato 0,2 mol/L (pH 9 y ácido linoleico (7,8-250 mmol/L como sustrato, ensayándose muestras paralelas incubadas con Tween-20/H2O (2 % (vehículo, D-003 (0,6-6 000 µg/mL o extracto de Perna canaliculus (50 µg/mL (sustancia de referencia. Se evaluó la actividad enzimática mediante el cambio de absorbancia a 234 nm producido por la formación de dienos conjugados y medido en espectrofotómetro UV-visible. Resultados: la adición de D-003 produjo una inhibición dosis dependiente de la actividad enzimática de la 5-LOX (r= 0,975; p< 0,05 (CI50= 23,06 µg/mL in vitro. La magnitud de esta inhibición fue moderada, ya que la inhibición máxima, alcanzada a partir de 1 250 µg/mL, resultó de solo un 30 %. Conclusiones: el estudio demuestra que el D-003 es capaz de inhibir la actividad enzimática de la 5-LOX in vitro, pero moderadamente.

Metabolite Damage and Metabolite Damage Control in Plants

Energy Technology Data Exchange (ETDEWEB)

Hanson, Andrew D. [Horticultural Sciences Department and; Henry, Christopher S. [Mathematics and Computer Science Division, Argonne National Laboratory, Argonne, Illinois 60439, email:; Computation Institute, University of Chicago, Chicago, Illinois 60637; Fiehn, Oliver [Genome Center, University of California, Davis, California 95616, email:; de Crécy-Lagard, Valérie [Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611, email: ,

2016-04-29

It is increasingly clear that (a) many metabolites undergo spontaneous or enzyme-catalyzed side reactions in vivo, (b) the damaged metabolites formed by these reactions can be harmful, and (c) organisms have biochemical systems that limit the buildup of damaged metabolites. These damage-control systems either return a damaged molecule to its pristine state (metabolite repair) or convert harmful molecules to harmless ones (damage preemption). Because all organisms share a core set of metabolites that suffer the same chemical and enzymatic damage reactions, certain damage-control systems are widely conserved across the kingdoms of life. Relatively few damage reactions and damage-control systems are well known. Uncovering new damage reactions and identifying the corresponding damaged metabolites, damage-control genes, and enzymes demands a coordinated mix of chemistry, metabolomics, cheminformatics, biochemistry, and comparative genomics. This review illustrates the above points using examples from plants, which are at least as prone to metabolite damage as other organisms.

Thiopurine methyltransferase genotype-phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia.

Science.gov (United States)

Lennard, Lynne; Cartwright, Cher Suzanne; Wade, Rachel; Richards, Susan M; Vora, Ajay

2013-07-01

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

CRISPR/Cas9-mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre-loxP-mediated lineage tracing.

Science.gov (United States)

Ma, Yuanwu; Yu, Lei; Pan, Shuo; Gao, Shan; Chen, Wei; Zhang, Xu; Dong, Wei; Li, Jing; Zhou, Rui; Huang, Lan; Han, Yunlin; Bai, Lin; Zhang, Li; Zhang, Lianfeng

2017-10-01

The rat is an important laboratory animal for physiological, toxicological and pharmacological studies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) is a simple and efficient tool to generate precise genetic modifications in rats, which will promote the accumulation of genetic resources and enable more precise studies of gene function. To monitor Cre-loxP-mediated excision in vivo, we generated a Cre reporter rat strain (Rosa26-imCherry) by knockin of a Cre reporter cassette at the Rosa26 locus using CRISPR/Cas9. Rosa26-imCherry rats exhibited inducible expression of the mCherry cassette (imCherry) using the Cre-loxP system, whereas normal rats exhibited ubiquitous expression of eGFP but not mCherry in the whole body. Injection of adeno-associated virus serotype 9-Cre into the hippocampus and skeletal muscle resulted in mCherry expression in virus-infected cells. Cre-loxP-mediated mCherry expression was then evaluated by crossing Rosa26-imCherry rats with transgenic rats ubiquitously expressing CAG-Cre, heart-specific α-MHC-Cre transgenic rats and liver-specific Alb-Cre knockin rats. Finally, using the established system the expression pattern of Cre driven by two endogenous gene promoters (Wfs1-Cre knockin rat, FabP2-Cre knockin rat) was traced. In summary, we demonstrated excision of the loxP-flanked allele in Rosa26-imCherry rats via activation of mCherry expression in the presence of Cre recombinase. This newly established Rosa26-imCherry rat strain represents a useful tool to facilitate Cre-expression pattern determination and tracing experiments. © 2017 Federation of European Biochemical Societies.

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism.

Science.gov (United States)

Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R

2009-01-01

Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.

A non-specific effect associated with conditional transgene expression based on Cre-loxP strategy in mice.

Directory of Open Access Journals (Sweden)

Linghua Qiu

Full Text Available Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.

Studying inhibition of calcium oxalate stone formation: an in vitro approach for screening hydrogen sulfide and its metabolites

Directory of Open Access Journals (Sweden)

S. Vaitheeswari

2015-06-01

Full Text Available ABSTRACTPurpose:Calcium oxalate urolithiasis is one of the most common urinary tract diseases and is of high prevalence. The present study proposes to evaluate the antilithiatic property of hydrogen sulfide and its metabolites like thiosulfate & sulfate in an in vitro model.Materials and Methods:The antilithiatic activity of sodium hydrogen sulfide (NaSH, sodium thiosulfate (Na2S2O3 and sodium sulfate (Na2SO4 on the kinetics of calcium oxalate crystal formation was investigated both in physiological buffer and in urine from normal and recurrent stone forming volunteers. The stones were characterized by optical and spectroscopic techniques.Results:The stones were characterized to be monoclinic, prismatic and bipyramidal habit which is of calcium monohydrate and dihydrate nature. The FTIR displayed fingerprint corresponding to calcium oxalate in the control while in NaSH treated, S=O vibrations were visible in the spectrum. The order of percentage inhibition was NaSH>Na2S2O3>Na2SO4.Conclusion:Our study indicates that sodium hydrogen sulfide and its metabolite thiosulfate are inhibitors of calcium oxalate stone agglomeration which makes them unstable both in physiological buffer and in urine. This effect is attributed to pH changes and complexing of calcium by S2O32-and SO42- moiety produced by the test compounds.

LOX/LH2 propulsion system for launch vehicle upper stage, test results

Science.gov (United States)

Ikeda, T.; Imachi, U.; Yuzawa, Y.; Kondo, Y.; Miyoshi, K.; Higashino, K.

1984-01-01

The test results of small LOX/LH2 engines for two propulsion systems, a pump fed system and a pressure fed system are reported. The pump fed system has the advantages of higher performances and higher mass fraction. The pressure fed system has the advantages of higher reliability and relative simplicity. Adoption of these cryogenic propulsion systems for upper stage of launch vehicle increases the payload capability with low cost. The 1,000 kg thrust class engine was selected for this cryogenic stage. A thrust chamber assembly for the pressure fed propulsion system was tested. It is indicated that it has good performance to meet system requirements.

Organophosphate esters in East Greenland polar bears and ringed seals: Adipose tissue concentrations and in vitro depletion and metabolite formation.

Science.gov (United States)

Strobel, Adelle; Willmore, William G; Sonne, Christian; Dietz, Rune; Letcher, Robert J

2018-04-01

East Greenland is a contamination "hot spot" for long-range transported anthropogenic chemicals, including organophosphate esters (OPEs). High concentrations of OPEs have been reported in arctic air while very little is known for wildlife where OPE tissue residues levels appear to be strongly influenced by biotransformation. In the present study, the hepatic in vitro metabolism of six environmentally relevant organophosphate (OP) triesters and corresponding OP diester formation were investigated in East Greenland polar bears (PBs) and ringed seals (RSs). The in vitro metabolism assay results were compared to adipose levels in field samples from the same individuals. In vitro OP triester metabolism was generally rapid and structure-dependent, where PBs metabolized OPEs more rapidly than RSs. Exceptions were the lack of triethyl phosphate (TEP) metabolism and slow metabolism of tris(2-ethylhexyl) phosphate (TEHP) in both species. OP diester metabolites were also formed with the exception of TEP which was not metabolized at all. Tris(1,3-dichloro-2-propyl) phosphate was completely converted to its corresponding diester. However, the mass balances showed that OP diester formation corresponding to TEHP, tri(n-butyl) phosphate, and tris(2-butyoxyethyl) phosphate did not account for 100% of the OP triester depletion, which indicated alternate pathways of OP triester metabolism had occurred. Triphenyl phosphate was completely converted to its OP diester metabolite in PBs but not in RSs suggesting species-specific differences. The results demonstrated that OP triester bioaccumulation and fate in PBs versus their RS prey is substantially influenced by biotransformation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells.

Science.gov (United States)

Kim, Jong-Sik; Baek, Seung Joon; Bottone, Frank G; Sali, Tina; Eling, Thomas E

2005-09-01

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

Science.gov (United States)

Wu, Xianai; Pramanik, Ananya; Duffel, Michael W.; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim; Kania-Korwel, Izabela

2011-01-01

Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of

12/15 lipoxygenase regulation of colorectal tumorigenesis is determined by the relative tumor levels of its metabolite 12-HETE and 13-HODE in animal models

OpenAIRE

Chang, Jian; Jiang, Li; Wang, Yinqiu; Yao, Bing; Yang, Shilin; Zhang, Bixiang; Zhang, Ming-Zhi

2014-01-01

Colorectal cancer (CRC) continues to be a major cause of morbidity and mortality. The arachidonic acid (AA) pathway and linoleic acid (LA) pathway have been implicated as important contributors to CRC development and growth. Human 15-lipoxygenase 1 (15-LOX-1) converts LA to anti-tumor 13-S-hydroxyoctadecadienoic acid (13-HODE)and 15-LOX-2 converts AA to 15-hydroxyeicosatetraenoic acid (15-HETE). In addition, human 12-LOX metabolizes AA to pro-tumor 12-HETE. In rodents, the function of 12-LOX ...

Benzene: a case study in parent chemical and metabolite interactions.

Science.gov (United States)

Medinsky, M A; Kenyon, E M; Schlosser, P M

1995-12-28

Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia and pancytopenia, and acute myelogenous leukemia. A combination of metabolites (hydroquinone and phenol for example) is apparently necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Since benzene and its hydroxylated metabolites (phenol, hydroquinone and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus, the potential exists for competition among various enzymes for phenol. However, zonal localization of Phase I and Phase II enzymes in various regions of the liver acinus regulates this competition. Biologically-based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.

Biotransformation of cannabidiol in mice. Identification of new acid metabolites.

Science.gov (United States)

Martin, B R; Harvey, D J; Paton, W D

1977-01-01

The in vivo metabolism of cannabidiol (CBD) was investigated in mice. Following the ip administration of CBD to mice, livers were removed and metabolites were extracted with ethyl acetate prior to partial purification on Sephadex LH-20 columns. Fractions from the columns were converted into trimethylsilyl, d9-trimethylsilyl, and methylester-trimethylsilyl derivatives for analysis by gas-liquid chromatography-mass spectrometry. In addition, metabolites containing carboxylic acid and ketone functional groups were reduced to alcohols with lithium aluminum deuteride before trimethylsilation. A total of 22 metabolites were characterized, 14 of which had not been reported previously. The metabolites could be categorized as follows: monohydroxylated (N=2), dihydroxylated (N=3), CBD-7-oic acid, side chain hydroxy-GBD-7-oic acids (N=3), side-chain acids (N=3), 7-hydroxy-side-chain acids (N=4), 6-oxo-side-chain acids (N=3) and glucuronide conjugates (N=3). The most significant biotransformations were glucuronide conjugation and, to a lesser extent, formation of CBD-7-oic acid.

The 5-lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines.

Science.gov (United States)

Loftus, J P; Cavatorta, D; Bushey, J J; Levine, C B; Sevier, C S; Wakshlag, J J

2016-06-01

The 5-lipoxygenase (5-LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase-3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5-LOX inhibitor regardless of cellular 5-LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5-LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide-3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response. © 2014 John Wiley & Sons Ltd.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

Science.gov (United States)

Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

2016-04-15

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of Volatile Metabolites of Garlic in Human Breast Milk

Science.gov (United States)

Scheffler, Laura; Sauermann, Yvonne; Zeh, Gina; Hauf, Katharina; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea

2016-01-01

The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography−mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO2 are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences. PMID:27275838

Nitric oxide metabolites during anoxia and reoxygenation in the anoxia-tolerant vertebrate Trachemys scripta

DEFF Research Database (Denmark)

Jensen, Frank Bo; Hansen, Marie Niemann; Montesanti, Gabriella

2014-01-01

this mechanism by up-regulating nitrite and other nitrite/NO metabolites (S-nitroso and iron-nitrosyl compounds) in several tissues when exposed to anoxia. We investigated whether this is a common strategy amongst anoxia-tolerant vertebrates by evaluating NO metabolites in red-eared slider turtles during long......-regulation of nitrite and other NO metabolites could be a general cytoprotective strategy amongst anoxia-tolerant vertebrates. The possible mechanisms of nitrite-derived NO and S-nitrosation in protecting cells from destructive Ca2+ influx during anoxia and in limiting ROS formation during reoxygenation are discussed....

Role of amino acid metabolites in the formation of soil organic matter

DEFF Research Database (Denmark)

Sørensen, Lasse Holst

1972-01-01

Carbon-14 labelled cellulose or glucose were added to a medium loam and two sandy soils. The soils were incubated at 20°C for about 6 yr under laboratory conditions. Six to 12 per cent of the labelled carbon added to the soils was transformed into metabolites hydrolysable to amino acids during th...

13C-NMR reveals glycerol as an unexpected major metabolite of the protozoan parasite Trichomonas vaginalis

International Nuclear Information System (INIS)

Chapman, A.; Lloyd, D.; Linstead, D.J.; Williams, J.

1985-01-01

13 C-NMR has been used to study the kinetics of the formation of metabolites from [l- 13 C]glucose in intact cells of Trichomonas vaginalis during anaerobic incubation. As well as the expected metabolites lactate and acetate, this technique revealed glycerol as an additional major product, present in amounts equimolar with acetate. The formation of glycerol is readily explained in terms of the need to maintain redox balance. This protozoan now joins the small group of organisms which are known to produce glycerol as a result of normal metabolic activities. (Auth.)

Prediction of metabolites of epoxidation reaction in MetaTox.

Science.gov (United States)

Rudik, A V; Dmitriev, A V; Bezhentsev, V M; Lagunin, A A; Filimonov, D A; Poroikov, V V

2017-10-01

Biotransformation is a process of the chemical modifications which may lead to the reactive metabolites, in particular the epoxides. Epoxide reactive metabolites may cause the toxic effects. The prediction of such metabolites is important for drug development and ecotoxicology studies. Epoxides are formed by some oxidation reactions, usually catalysed by cytochromes P450, and represent a large class of three-membered cyclic ethers. Identification of molecules, which may be epoxidized, and indication of the specific location of epoxide functional group (which is called SOE - site of epoxidation) are important for prediction of epoxide metabolites. Datasets from 355 molecules and 615 reactions were created for training and validation. The prediction of SOE is based on a combination of LMNA (Labelled Multilevel Neighbourhood of Atom) descriptors and Bayesian-like algorithm implemented in PASS software and MetaTox web-service. The average invariant accuracy of prediction (AUC) calculated in leave-one-out and 20-fold cross-validation procedures is 0.9. Prediction of epoxide formation based on the created SAR model is included as the component of MetaTox web-service ( http://www.way2drug.com/mg ).

Regulatory metabolites of vitamin E and their putative relevance for atherogenesis

Directory of Open Access Journals (Sweden)

Maria Wallert

2014-01-01

Full Text Available Vitamin E is likely the most important antioxidant in the human diet and α-tocopherol is the most active isomer. α-Tocopherol exhibits anti-oxidative capacity in vitro, and inhibits oxidation of LDL. Beside this, α-tocopherol shows anti-inflammatory activity and modulates expression of proteins involved in uptake, transport and degradation of tocopherols, as well as the uptake, storage and export of lipids such as cholesterol. Despite promising anti-atherogenic features in vitro, vitamin E failed to be atheroprotective in clinical trials in humans. Recent studies highlight the importance of long-chain metabolites of α-tocopherol, which are formed as catabolic intermediate products in the liver and occur in human plasma. These metabolites modulate inflammatory processes and macrophage foam cell formation via mechanisms different than that of their metabolic precursor α-tocopherol and at lower concentrations. Here we summarize the controversial role of vitamin E as a preventive agent against atherosclerosis and point the attention to recent findings that highlight a role of these long-chain metabolites of vitamin E as a proposed new class of regulatory metabolites. We speculate that the metabolites contribute to physiological as well as pathophysiological processes.

Close-in characteristics of LH2/LOX reactions

Science.gov (United States)

Riehl, W. A.; Ullian, L. J.

1985-01-01

In deriving shock overpressures from space vehicles employing LH2 and LOX, separate methods of analyses and prediction are recommended, as a function of the distance. Three methods of treatment are recommended. For the Far Field - where the expected shock overpressure is less than 40 psi (lambda = 5) - use the classical PYRO approach to determine TNT yield, and employ classical ordnance (Kingery) curve to obtain the overall value. For the Close-In Range, a suggested limit is 3D, or a zone from a distance of three times the tank diameter to the tank wall. Rather than estimate a specific distance from the center of explosion to the target, it is only necessary to estimate whether this could be within one, two, or three diameters away from the wall; i.e., in the 1, 2, or 3D zone. Then assess whether mixing mode is by the PYRO CBGS (spill) mode or CBM (internal mixing) mode. From the zone and mixing mode, the probability of attaining various shock overpressures is determined from the plots provided herein. For the transition zone, between 40 psi and the 3D distance, it is tentatively recommended that both of the preceding methods be used, and to be conservative, the higher resulting value be used.

Mechanisms of foam cell formation in atherosclerosis.

Science.gov (United States)

Chistiakov, Dimitry A; Melnichenko, Alexandra A; Myasoedova, Veronika A; Grechko, Andrey V; Orekhov, Alexander N

2017-11-01

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.

S.S.T.O. performance assessment with in-flight lox collection

Science.gov (United States)

Vandenkerckhove, J.; Czysz, P.

1995-10-01

Much attention has recently been given, up to harware development to in-flight oxygen collection as a means to improve considerably the performance of both TSTO & SSTO vehicles. A first assessment suggests that it permits simultaneously to improve much both gross take-off weight (by more than 30%) & dry weight (by more than 15%) of an SSTO and to lower significantly the Mach number of transition scramjet → rocket, from 15 down below 10, thereby reducing dramatically the programmatic development risks. After having compared in-flight lox collection with other SSTO concepts, this paper provides a tentative assessment of the performance of SSTO vehicles taking advantage of it, in particular their sensitivity to changes in system characteristics such as transition Mach number, vehicle slenderness (i.e. Küchemann's parameter τ) or planform loading at take-off and in collection characteristics, in particular collection ratio & specific collection plant weight.

A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

Science.gov (United States)

Kawano, Fuun; Okazaki, Risako; Yazawa, Masayuki; Sato, Moritoshi

2016-12-01

Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m -2 ) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

Science.gov (United States)

Wong, Timothy; Wang, Zhican; Chapron, Brian D; Suzuki, Mizuki; Claw, Katrina G; Gao, Chunying; Foti, Robert S; Prasad, Bhagwat; Chapron, Alenka; Calamia, Justina; Chaudhry, Amarjit; Schuetz, Erin G; Horst, Ronald L; Mao, Qingcheng; de Boer, Ian H; Thornton, Timothy A; Thummel, Kenneth E

2018-04-01

Metabolism of 25-hydroxyvitamin D 3 (25OHD 3 ) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD 3 has been extensively investigated, limited information is available on the conjugation of 25OHD 3 In this study, we report that 25OHD 3 is selectively conjugated to 25OHD 3 -3- O -sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD 3 , 25OHD 3 -3- O -sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD 3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD 3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD 3 -3- O -sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD 3 -3- O -sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD 3 -3- O -sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD 3 in vivo, and contribute indirectly to the biologic effects of vitamin D. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Metabolism of metofluthrin in rats: I. Identification of metabolites.

Science.gov (United States)

Abe, Jun; Nagahori, Hirohisa; Tarui, Hirokazu; Tomigahara, Yoshitaka; Isobe, Naohiko

2018-02-01

1. Metofluthrin (2,3,5,6-tetrafluoro-4-(methoxymethyl)benzyl (Z/E)-(1R)-trans-2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylate) is a novel pyrethroid insecticide, which has E/Z isomers at prop-1-enyl group. 2. Rats were orally dosed with each [ 14 C]-labelled E/Z isomer, and the excreta were collected for isolation and identification of metabolites. Analysis of the excreta by LC/MS and NMR revealed formation of 33 and 23 (total 42) metabolites from rats dosed with Z-isomer and E-isomer, respectively. 3. Major metabolic reactions were cleavage of ester linkage, O-demethylation, hydroxylation, epoxidation or reduction of double bond, glutathione conjugation and its further metabolism, hydroxylation of epoxide and formation of lactone ring. Notably, the acid side, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, was much more variously metabolised compared to chrysanthemic acid, the acid side of the known pyrethroids. 4. Major metabolites for Z-isomer mostly retained ester linkage with 1,2-dihydroxypropyl group and/or 2-methylalcohol of cyclopropane ring, while most of those for E-isomer received hydrolysis of the ester linkage without oxidation at the 1-propenyl group or the gem-methyl groups, suggesting epoxidation and hydroxylation could occur more easily on Z-isomer. 5. As the novel metabolic pathways for pyrethroids, isomerisation of ω-carboxylic acid moiety, reduction or hydration of double bond and cleavage of cyclopropane ring via epoxidation were suggested.

Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome.

Science.gov (United States)

Busnadiego, O; Gorbenko Del Blanco, D; González-Santamaría, J; Habashi, J P; Calderon, J F; Sandoval, P; Bedja, D; Guinea-Viniegra, J; Lopez-Cabrera, M; Rosell-Garcia, T; Snabel, J M; Hanemaaijer, R; Forteza, A; Dietz, H C; Egea, G; Rodriguez-Pascual, F

2015-08-01

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-β (TGF-β)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1(C1039G/+)) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1(mgR/mgR), without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-β signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-β signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1(C1039G/+) mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS. Copyright © 2015 Elsevier

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells

Directory of Open Access Journals (Sweden)

Peng Liu

2018-05-01

Full Text Available Sulforaphane (SFN exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane-N-acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H2O2 challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2-antioxidant response element (ARE pathway and the induction of intracellular glutathione (GSH played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

A new paradigm for known metabolite identification in metabonomics/metabolomics: metabolite identification efficiency.

Science.gov (United States)

Everett, Jeremy R

2015-01-01

A new paradigm is proposed for assessing confidence in the identification of known metabolites in metabonomics studies using NMR spectroscopy approaches. This new paradigm is based upon the analysis of the amount of metabolite identification information retrieved from NMR spectra relative to the molecular size of the metabolite. Several new indices are proposed including: metabolite identification efficiency (MIE) and metabolite identification carbon efficiency (MICE), both of which can be easily calculated. These indices, together with some guidelines, can be used to provide a better indication of known metabolite identification confidence in metabonomics studies than existing methods. Since known metabolite identification in untargeted metabonomics studies is one of the key bottlenecks facing the science currently, it is hoped that these concepts based on molecular spectroscopic informatics, will find utility in the field.

A New Paradigm for Known Metabolite Identification in Metabonomics/Metabolomics: Metabolite Identification Efficiency

Directory of Open Access Journals (Sweden)

Jeremy R. Everett

2015-01-01

Full Text Available A new paradigm is proposed for assessing confidence in the identification of known metabolites in metabonomics studies using NMR spectroscopy approaches. This new paradigm is based upon the analysis of the amount of metabolite identification information retrieved from NMR spectra relative to the molecular size of the metabolite. Several new indices are proposed including: metabolite identification efficiency (MIE and metabolite identification carbon efficiency (MICE, both of which can be easily calculated. These indices, together with some guidelines, can be used to provide a better indication of known metabolite identification confidence in metabonomics studies than existing methods. Since known metabolite identification in untargeted metabonomics studies is one of the key bottlenecks facing the science currently, it is hoped that these concepts based on molecular spectroscopic informatics, will find utility in the field.

Fate of N-methylformamide in mice. Routes of elimination and characterization of metabolites

International Nuclear Information System (INIS)

Kestell, P.; Gescher, A.; Slack, J.A.

1985-01-01

The fate of N-methylformamide has been investigated in male CBA/CA mice following the administration of this compound labeled with 14 C either in the methyl or in the formyl group. The major route of elimination was found to be via the kidneys although a substantial quantity (39% of the dose) was eliminated via the lungs as CO 2 in the case of [ 14 C]formyl-labeled N-methylformamide. In addition to the unchanged compound three metabolites were found in the urine by TLC autoradiography. One of these metabolites was identified as methylamine after conversion to its 2,4-dinitrophenyl derivative. The derivative was isolated and shown to be N-methyl-2,4-dinitroaniline by mass spectrometry. Further evidence that methylamine was a metabolite of N-methylformamide was provided by ion pair HPLC analysis of urine from mice dosed with [ 14 C]methyl-labeled N-methylformamide. The second metabolite was tentatively identified as N-hydroxymethylformamide which was present in the urine of mice dosed with either [ 14 C]methyl- or [ 14 C]formyl-labeled N-methylformamide. Formate was not a urinary metabolite of N-methylformamide. The identity of the third urinary metabolite remains unknown

Double di oxygenation by mouse 8S-lipoxygenase: Specific formation of a potent peroxisome proliferator-activated receptor α agonist

International Nuclear Information System (INIS)

Jisaka, Mitsuo; Iwanaga, Chitose; Takahashi, Nobuyuki; Goto, Tsuyoshi; Kawada, Teruo; Yamamoto, Tatsuyuki; Ikeda, Izumi; Nishimura, Kohji; Nagaya, Tsutomu; Fushiki, Tohru; Yokota, Kazushige

2005-01-01

Mouse 8S-lipoxygenase (8-LOX) metabolizes arachidonic acid (AA) specifically to 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE), which will be readily reduced under physiological circumstances to 8S-hydroxyeicosatetraenoic acid (8S-HETE), a natural agonist of peroxisome proliferator-activated receptor α (PPARα). Here, we investigated whether 8-LOX could further oxygenate AA and whether the products could activate PPARs. The purified recombinant 8-LOX converted AA exclusively to 8S-HPETE and then to (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid (8S,15S-diHPETE). The k cat /K m values for 8S-HPETE and AA were 3.3 x 10 3 and 2.7 x 10 4 M -1 s -1 , respectively. 8-LOX also dioxygenated 8S-HETE and 15S-H(P)ETE specifically to the corresponding 8S,15S-disubstituted derivatives. By contrast, 15-LOX-2, a human homologue of 8-LOX, produced 8S,15S-diH(P)ETE from 8S-H(P)ETE but not from AA nor 15S-H(P)ETE. 8S,15S-diHETE activated PPARα more strongly than 8S-HETE did. The present results suggest that 8S,15S-diH(P)ETE as well as 8S-H(P)ETE would contribute to the physiological function of 8-LOX and also that 8-LOX can function as a potential 15-LOX

Evaluation of Olive Fruit Lipoxygenase Extraction Protocols on 9- and 13-Z,E-HPODE Formation

Directory of Open Access Journals (Sweden)

Barbara Soldo

2016-04-01

Full Text Available In plant tissues, enzymes implicated in the lipoxygenase (LOX pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.

Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae.

Science.gov (United States)

Son, Eun Yeong; Lee, Sang Mi; Kim, Minjoo; Seo, Jeong-Ah; Kim, Young-Suk

2018-07-01

This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methylbenzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and non-volatile metabolites in rice wine, facilitating their use in brewing rice wine. Copyright © 2018. Published by Elsevier Ltd.

Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon

DEFF Research Database (Denmark)

Autrup, Herman

1979-01-01

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation......-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon....

Metabolomic homeostasis shifts after callus formation and shoot regeneration in tomato

Science.gov (United States)

Kumari, Alka; Ray, Kamalika; Sadhna, Sadhna; Pandey, Arun Kumar; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

2017-01-01

Plants can regenerate from a variety of tissues on culturing in appropriate media. However, the metabolic shifts involved in callus formation and shoot regeneration are largely unknown. The metabolic profiles of callus generated from tomato (Solanum lycopersicum) cotyledons and that of shoot regenerated from callus were compared with the pct1-2 mutant that exhibits enhanced polar auxin transport and the shr mutant that exhibits elevated nitric oxide levels. The transformation from cotyledon to callus involved a major shift in metabolite profiles with denser metabolic networks in the callus. In contrast, the transformation from callus to shoot involved minor changes in the networks. The metabolic networks in pct1-2 and shr mutants were distinct from wild type and were rewired with shifts in endogenous hormones and metabolite interactions. The callus formation was accompanied by a reduction in the levels of metabolites involved in cell wall lignification and cellular immunity. On the contrary, the levels of monoamines were upregulated in the callus and regenerated shoot. The callus formation and shoot regeneration were accompanied by an increase in salicylic acid in wild type and mutants. The transformation to the callus and also to the shoot downregulated LST8 and upregulated TOR transcript levels indicating a putative linkage between metabolic shift and TOR signalling pathway. The network analysis indicates that shift in metabolite profiles during callus formation and shoot regeneration is governed by a complex interaction between metabolites and endogenous hormones. PMID:28481937

Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid.

Science.gov (United States)

Isobe, Yosuke; Kawashima, Yusuke; Ishihara, Tomoaki; Watanabe, Kenji; Ohara, Osamu; Arita, Makoto

2018-04-20

The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

Science.gov (United States)

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Is cardiovascular disease in patients with diabetes associated with serum levels of MMP-2, LOX, and the elastin degradation products ELM and ELM-2?

Science.gov (United States)

Preil, Simone Andrea Rørdam; Thorsen, Anne-Sofie Faarvang; Christiansen, Anne Lindegaard; Poulsen, Mikael Kjær; Karsdal, Morten Asser; Leeming, Diana Julie; Rasmussen, Lars Melholt

2017-11-01

Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression in diabetic patients, namely matrix metalloproteinase 2 (MMP-2), lysyl oxidase (LOX) and elastin itself. In this study we investigate whether the serum concentrations of elastin-related proteins correlate to signs of CVD in patients with T2DM. Blood samples from 302 type 2 diabetic patients were analysed for MMP-2, LOX, and the elastin degradation products ELM and ELM2. The results were investigated for correlations to signs of CVD in different vascular territories, as determined by myocardial perfusion scintigraphy, carotid artery thickness and ankle-brachial blood pressure index. T2DM patients with peripheral arterial disease (low ankle-brachial index) (PAD) display higher levels of MMP-2 and ELM compared to patients without PAD. However, none of the proteins or degradation products correlated with myocardial ischemia or a combined measure of CVD-signs, including myocardial ischemia, increased carotid thickness and decreased ankle-brachial blood pressure. Our results suggest that the diabetic environment affects the circulating amounts of MMP-2 and ELM in patients with PAD. However, the same connection could not be seen in diabetic patients with CVD broadly identified in three vascular territories. LOX and ELM-2 did not correlate to any type of CVD. Overall, serum levels of elastin-related molecules are only remotely related to CVD in type 2 diabetes.

Regional gene expression of LOX-1, VCAM-1, and ICAM-1 in aorta of HIV-1 transgenic rats.

Directory of Open Access Journals (Sweden)

Anne Mette Fisker Hag

Full Text Available BACKGROUND: Increased prevalence of atherosclerotic cardiovascular disease in HIV-infected patients has been observed. The cause of this accelerated atherosclerosis is a matter of controversy. As clinical studies are complicated by a multiplicity of risk-factors and a low incidence of hard endpoints, studies in animal models could be attractive alternatives. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1, vascular cell adhesion molecule-1 (VCAM-1, and intercellular adhesion molecule-1 (ICAM-1 in HIV-1 transgenic (HIV-1Tg rats; these genes are all thought to play important roles in early atherogenesis. Furthermore, the plasma level of sICAM-1 was measured. We found that gene expressions of LOX-1 and VCAM-1 were higher in the aortic arch of HIV-1Tg rats compared to controls. Also, the level of sICAM-1 was elevated in the HIV-1Tg rats compared to controls, but the ICAM-1 gene expression profile did not show any differences between the groups. CONCLUSIONS/SIGNIFICANCE: HIV-1Tg rats have gene expression patterns indicating endothelial dysfunction and accelerated atherosclerosis in aorta, suggesting that HIV-infection per se may cause atherosclerosis. This transgenic rat model may be a very promising model for further studies of the pathophysiology behind HIV-associated cardiovascular disease.

MicroRNA-98 rescues proliferation and alleviates ox-LDL-induced apoptosis in HUVECs by targeting LOX-1

Science.gov (United States)

Chen, Zhibo; Wang, Mian; He, Qiong; Li, Zilun; Zhao, Yang; Wang, Wenjian; Ma, Jieyi; Li, Yongxin; Chang, Guangqi

2017-01-01

Oxidized low-density lipoprotein (ox-LDL) is a major and critical mediator of atherosclerosis, and the underlying mechanism is thought to involve the ox-LDL-induced dysfunction of endothelial cells (ECs). MicroRNAs (miRNAs), which are a group of small non-coding RNA molecules that post-transcriptionally regulate the expression of target genes, have been associated with diverse cellular functions and the pathogenesis of various diseases, including atherosclerosis. miRNA-98 (miR-98) has been demonstrated to be involved in the regulation of cellular apoptosis; however, the role of miR-98 in ox-LDL-induced dysfunction of ECs and atherosclerosis has yet to be elucidated. Therefore, the present study aimed to investigate the role of miR-98 in ox-LDL-induced dysfunction of ECs and the underlying mechanism. It was demonstrated that miR-98 expression was markedly downregulated in ox-LDL-treated human umbilical vein ECs (HUVECs) and that miR-98 promoted the proliferation and alleviated apoptosis of HUVECs exposed to ox-LDL. In addition, the results demonstrated that lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) was a direct target of miR-98 in HUVECs, as indicated by a luciferase assay. The results of the present study suggested that miR-98 may inhibit the uptake of toxic ox-LDL, maintain HUVEC proliferation and protect HUVECs against apoptosis via the suppression of LOX-1. PMID:28565756

PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

Directory of Open Access Journals (Sweden)

A.M. NOSOV

2014-06-01

Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures. 

Characterization of model peptide adducts with reactive metabolites of naphthalene by mass spectrometry.

Directory of Open Access Journals (Sweden)

Nathalie T Pham

Full Text Available Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.

LC-MS-MS identification of drug metabolites obtained by metalloporphyrin mediated oxidation

Directory of Open Access Journals (Sweden)

Maurin Andrea J. M.

2003-01-01

Full Text Available In this paper we report the application of liquid chromatography-mass spectrometry (LC-MS-MS to the identification of the products formed by oxidation of albendazole and disopyramide with metalloporphyrins in dichloroethane, using iodosylbenzene as an oxygen donor. Our results show that LC-MS-MS is a powerful tool to study the in vitro metabolism of drugs, allowing the identification of known and unknown metabolites. In addition, it was observed that the catalyst system used resulted in the formation of the same metabolites as obtained in vivo, although for disopyramide other products were also observed.

Complicating factors in safety testing of drug metabolites: Kinetic differences between generated and preformed metabolites

International Nuclear Information System (INIS)

Prueksaritanont, Thomayant; Lin, Jiunn H.; Baillie, Thomas A.

2006-01-01

This paper aims to provide a scientifically based perspective on issues surrounding the proposed toxicology testing of synthetic drug metabolites as a means of ensuring adequate nonclinical safety evaluation of drug candidates that generate metabolites considered either to be unique to humans or are present at much higher levels in humans than in preclinical species. We put forward a number of theoretical considerations and present several specific examples where the kinetic behavior of a preformed metabolite given to animals or humans differs from that of the corresponding metabolite generated endogenously from its parent. The potential ramifications of this phenomenon are that the results of toxicity testing of the preformed metabolite may be misleading and fail to characterize the true toxicological contribution of the metabolite when formed from the parent. It is anticipated that such complications would be evident in situations where (a) differences exist in the accumulation of the preformed versus generated metabolites in specific tissues, and (b) the metabolite undergoes sequential metabolism to a downstream product that is toxic, leading to differences in tissue-specific toxicity. Owing to the complex nature of this subject, there is a need to treat drug metabolite issues in safety assessment on a case-by-case basis, in which a knowledge of metabolite kinetics is employed to validate experimental paradigms that entail administration of preformed metabolites to animal models

Characterization of D-3-hydroxybutyrylcarnitine (ketocarnitine): an identified ketosis-induced metabolite

NARCIS (Netherlands)

Soeters, Maarten R.; Serlie, Mireille J.; Sauerwein, Hans P.; Duran, Marinus; Ruiter, Jos P.; Kulik, Willem; Ackermans, Mariëtte T.; Minkler, Paul E.; Hoppel, Charles L.; Wanders, Ronald J. A.; Houten, Sander M.

2012-01-01

Hydroxybutyrylcarnitine (HB-carnitine) is a metabolite that has been associated with insulin resistance and type 2 diabetes mellitus. It is currently unknown whether HB-carnitine can be produced from D-3-hydroxybutyrate (D-3HB), a ketone body; but its formation from L-3-HB-CoA, a fatty acid

Anti-Inflammatory Effects of Benfotiamine are Mediated Through the Regulation of Arachidonic Acid Pathway in Macrophages

Science.gov (United States)

Shoeb, Mohammad; Ramana, Kota V

2011-01-01

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent anti-oxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study indicates a novel role of benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating the arachidonic acid (AA) pathway generated inflammatory lipid mediators in RAW 264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes (LTB4), prostaglandin E2 (PGE2), thromboxanes 2 (TXB2) and prostacyclin (PGI2) in macrophages. Further, LPS-induced expressions of AA metabolizing enzymes such as COX-2, LOX-5, TXB synthase and PGI2 synthase were significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-kB, and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adducts formation. Most importantly, as compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented the LPS-induced macrophage death and monocytes adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. PMID:22067901

Mutagenic azide metabolite is azidoalanine

International Nuclear Information System (INIS)

Owais, W.M.; Rosichan, J.L.; Ronald, R.C.; Kleinhofs, A.; Nilan, R.A.

1981-01-01

Sodium axide produces high mutation rates in a number of species. Azide mutagenicity is mediated through a metabolite in barley and bacteria. Many studies showed that azide affects the L-cysteine biosynthesis pathway. Cell-free extracts of Salmonella typhimurium convert azide and O-acetylserine to the mutagenic metabolite. O-acetylserine sulfhydrylase was identified as the enzyme responsible for the metabolite biosynthesis. To confirm the conclusion that the azide metabolite is formed through the β-substitution pathway of L-cysteine, we radioactively labeled the azide metabolite using 14 C-labeled precursors. Moreover, the mutagenic azide metabolite was purified and identified as azidoalanine based on mass spectroscopy and elemental analysis. 26 refs., 3 figs., 1 tab

Caffeine metabolites not caffeine protect against riboflavin photosensitized oxidative damage related to skin and eye health

DEFF Research Database (Denmark)

Scurachio, R. S.; Mattiucci, F.; Santos, W. G.

2016-01-01

. Caffeine metabolites rather than caffeine seem accordingly important for the observed protective effect against cutaneous melanoma identified for drinkers of regular but not of decaffeinated coffee. The caffeine metabolites, but not caffeine, were by time resolved single photon counting found to quench...... singlet excited riboflavin through exothermic formation of ground-state precursor complexes indicating importance of hydrogen bounding through keto-enol tautomer's for protection of oxidizable substrates and sensitive structures against riboflavin photosensitization....

Cre/loxP-mediated adenovirus type 5 packaging signal excision demonstrates that core element VI is sufficient for virus packaging

International Nuclear Information System (INIS)

Maeda, Yasushi; Kimura, En; Uchida, Yuji; Nishida, Yasuto; Yamashita, Satoshi; Arima, Toshiyuki; Uchino, Makoto

2003-01-01

Previous analyses have demonstrated that packaging of the adenovirus type 5 (Ad5) genome is dependent on at least seven cis-acting elements, called AI to AVII, which are located in the left-end region of the genome. These elements have different packaging efficiencies, and without AI through AV, viral DNA cannot be packaged. Here we report the identification of the cis-acting Ad5 packaging domain in vivo by using the Cre/loxP system. We found that an adenoviral DNA fragment (nt 192 to nt 358), which includes elements AI to AV, is excised by Cre recombinase and packaged into capsids. Furthermore, this mutant adenovirus replicated so efficiently by repetitive propagation that its purification by CsCI equilibrium gradient was possible. This study clarified that the region from nt 358 to nt 454 on the viral genome is sufficient for packaging. Recently, the helper-dependent adenoviral vector (HDAd) construction system has been developed for the purpose of gene therapy. This system uses a helper virus with two parallel loxP sites flanking the packaging signal. This region is eliminated by Cre-mediated excision, which prevents helper virus packaging. Our data provide useful information regarding factors affecting efficient elimination

Focused library design and synthesis of 2-mercapto benzothiazole linked 1,2,4-oxadiazoles as COX-2/5-LOX inhibitors

Science.gov (United States)

Yatam, Satayanarayana; Gundla, Rambabu; Jadav, Surender Singh; Pedavenkatagari, Narayana reddy; Chimakurthy, Jithendra; Rani B, Namratha; Kedam, Thyagaraju

2018-05-01

Mercapto benzothiazole linked 1,2,4-oxadiazole derivatives were designed (4a-u) as new anti-inflammatory agents using bioisosteric approach and docking studies. The docking results clearly indicated that the compounds 4a-u shown good docking interaction towards COX-2 enzyme. In silico drug-like properties were also calculated for compounds (4a-u) and exhibited significant H-bond acceptor ratio. All compounds were synthesized and biologically evaluated using in vitro COX-1, COX-2 and 5-LOX assays. Compound 4k and 4q (IC50 = 6.8 μM and IC50 = 5.0 μM) found to be potent, selective COX-2 inhibitors and display better anti-inflammatory activity than standard Ibuprofen. Compound 4l and 4e found to be potent inhibitors against 5-LOX (IC50 = 5.1 μM and IC50 = 5.5 μM). The in vivo anti-inflammatory activity studies shown that the compounds 4q and 4k effectively reducing the paw edema volume at 3h and 5h than standard drug Ibuprofen. The DPPH radical scavenging activity provided anti-oxidant activity of compound 4e (IC50 = 25.6 μM) than reference standard Ascorbic acid.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study

Directory of Open Access Journals (Sweden)

Hermanova Marketa

2010-05-01

Full Text Available Abstract Background We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2 and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA and inhibitors of lipoxygenases (LOX and cyclooxygenases (COX. This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Methods Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Results Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2 or SH-SY5Y after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX in combination with ATRA in both cell lines. Conclusions Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Characterization of inter-tissue and inter-strain variability of TCE glutathione conjugation metabolites DCVG, DCVC, and NAcDCVC in the mouse.

Science.gov (United States)

Luo, Yu-Syuan; Furuya, Shinji; Chiu, Weihsueh; Rusyn, Ivan

2018-01-01

Trichloroethylene (TCE) is a ubiquitous environmental toxicant that is a liver and kidney carcinogen. Conjugation of TCE with glutathione (GSH) leads to formation of nepthrotoxic and mutagenic metabolites postulated to be critical for kidney cancerdevelopment; however, relatively little is known regarding their tissue levels as previous analytical methods for their detection lacked sensitivity. Here, an LC-MS/MS-based method for simultaneous detection of S-(1,2-dichlorovinyl)-glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC) in multiple mouse tissues was developed. This analytical method is rapid, sensitive (limits of detection (LOD) 3-30 fmol across metabolites and tissues), and robust to quantify all three metabolites in liver, kidneys, and serum. The method was used to characterize inter-tissue and inter-strain variability in formation of conjugative metabolites of TCE. Single oral dose of TCE (24, 240 or 800 mg/kg) was administered to male mice from 20 inbred strains of Collaborative Cross. Inter-strain variability in the levels of DCVG, DCVC, and NAcDCVC (GSD = 1.6-2.9) was observed. Whereas NAcDCVC was distributed equally among analyzed tissues, highest levels of DCVG were detected in liver and DCVC in kidneys. Evidence indicated that inter-strain variability in conjugative metabolite formation of TCE might affect susceptibility to adverse health effects and that this method might aid in filling data gaps in human health assessment of TCE.

Cold inducible promoter driven Cre-lox system proved to be highly efficient for marker gene excision in transgenic barley.

Science.gov (United States)

Éva, Csaba; Téglás, Flóra; Zelenyánszki, Helga; Tamás, Cecília; Juhász, Angéla; Mészáros, Klára; Tamás, László

2018-01-10

A Cre-lox based auto-excision strategy has been adapted for barley, capable of cre and selectable marker gene (SMG) removal. The cold inducible wheat promoter called wcs120 was utilised for driving Cre expression. The binary vector was carrying the transgene (uidA) and a so called 'recombination cassette' flanked by the lox sequences. This part included both the recombinase gene and the SMG (bar) under the control of a constitutive promoter. T 0 , T 1 and T 2 transgenic plants were subjected to low temperature (at 4°C, 10°C and 12°C) at different developmental stages to induce recombination. The presence of uidA, cre, and bar genes and recombination footprints were studied by PCR and DNA sequencing, while cre transcription was followed by qRT-PCR. These analyses indicated that, cold treatment of the germinating seeds (4°C for 3days) followed by plant growing at higher temperature (24°C) has been the most efficient (90-100%), and this treatment lead to heritable changes in the genome. Thermal separation of Cre accumulation (at low temperature) from Cre enzyme activity (at higher temperature) could have prevented the premature excision of its own encoding gene, and lead to high expression level thereby increasing recombination frequency. Copyright © 2017 Elsevier B.V. All rights reserved.

Immune regulation by microbiome metabolites.

Science.gov (United States)

Kim, Chang H

2018-03-22

Commensal microbes and the host immune system have been co-evolved for mutual regulation. Microbes regulate the host immune system, in part, by producing metabolites. A mounting body of evidence indicates that diverse microbial metabolites profoundly regulate the immune system via host receptors and other target molecules. Immune cells express metabolite-specific receptors such as P2X 7 , GPR41, GPR43, GPR109A, aryl hydrocarbon receptor precursor (AhR), pregnane X receptor (PXR), farnesoid X receptor (FXR), TGR5 and other molecular targets. Microbial metabolites and their receptors form an extensive array of signals to respond to changes in nutrition, health and immunological status. As a consequence, microbial metabolite signals contribute to nutrient harvest from diet, and regulate host metabolism and the immune system. Importantly, microbial metabolites bidirectionally function to promote both tolerance and immunity to effectively fight infection without developing inflammatory diseases. In pathogenic conditions, adverse effects of microbial metabolites have been observed as well. Key immune-regulatory functions of the metabolites, generated from carbohydrates, proteins and bile acids, are reviewed in this article. © 2018 John Wiley & Sons Ltd.

Effects of clopidogrel on the pharmacokinetics of sibutramine and its active metabolites.

Science.gov (United States)

Bae, Jung-Woo; Jang, Choon-Gon; Lee, Seok-Young

2011-12-01

Sibutramine is metabolized by the enzymes CYP2B6 and CYP2C19 into 2 active metabolites, M1 (mono-desmethyl sibutramine) and M2 (di-desmethyl sibutramine). Clopidogrel is a mechanism-based inhibitor of CYP2B6 and CYP2C19. In this study, 13 extensive metabolizers of CYP2B6 and CYP2C19 were evaluated to clarify whether clopidogrel inhibits the formation of the active metabolites of sibutramine. In the control phase, each subject received a 15-mg oral dose of sibutramine. After a washout period of 2 weeks, in the clopidogrel phase, the subjects received 300 mg of clopidogrel on the first day and then 75-mg once daily for 6 days. One hour after the last dosing of clopidogrel, all subjects received 15-mg of sibutramine. Compared with the control phase, the mean sibutramine and M1 plasma concentrations were higher after clopidogrel treatment. Clopidogrel significantly increased the half-life (242% of control phase) and area under the plasma concentration-time curve from 0 to infinity (AUC(inf)) (227% of control phase) of sibutramine and decreased the apparent oral clearance (31% of control phase) of sibutramine. Pharmacokinetic analysis showed significant increases in the AUC(inf) (162% of control phase) of M1. The CYP2B6 and CYP2C19 inhibitor clopidogrel significantly inhibited the formations of M1 from sibutramine and M2 from sibutramine by 37% and 64%, respectively. Therefore, CYP2B6 and CYP2C19 are in vivo catalysts for the formation of the 2 active metabolites of sibutramine.

New Methodology for Known Metabolite Identification in Metabonomics/Metabolomics: Topological Metabolite Identification Carbon Efficiency (tMICE).

Science.gov (United States)

Sanchon-Lopez, Beatriz; Everett, Jeremy R

2016-09-02

A new, simple-to-implement and quantitative approach to assessing the confidence in NMR-based identification of known metabolites is introduced. The approach is based on a topological analysis of metabolite identification information available from NMR spectroscopy studies and is a development of the metabolite identification carbon efficiency (MICE) method. New topological metabolite identification indices are introduced, analyzed, and proposed for general use, including topological metabolite identification carbon efficiency (tMICE). Because known metabolite identification is one of the key bottlenecks in either NMR-spectroscopy- or mass spectrometry-based metabonomics/metabolomics studies, and given the fact that there is no current consensus on how to assess metabolite identification confidence, it is hoped that these new approaches and the topological indices will find utility.

A Computational Method for Determining the Equilibrium Composition and Product Temperature in a LH2/LOX Combustor

Science.gov (United States)

Sozen, Mehmet

2003-01-01

In what follows, the model used for combustion of liquid hydrogen (LH2) with liquid oxygen (LOX) using chemical equilibrium assumption, and the novel computational method developed for determining the equilibrium composition and temperature of the combustion products by application of the first and second laws of thermodynamics will be described. The modular FORTRAN code developed as a subroutine that can be incorporated into any flow network code with little effort has been successfully implemented in GFSSP as the preliminary runs indicate. The code provides capability of modeling the heat transfer rate to the coolants for parametric analysis in system design.

Metabolite coupling in genome-scale metabolic networks

Directory of Open Access Journals (Sweden)

Palsson Bernhard Ø

2006-03-01

Full Text Available Abstract Background Biochemically detailed stoichiometric matrices have now been reconstructed for various bacteria, yeast, and for the human cardiac mitochondrion based on genomic and proteomic data. These networks have been manually curated based on legacy data and elementally and charge balanced. Comparative analysis of these well curated networks is now possible. Pairs of metabolites often appear together in several network reactions, linking them topologically. This co-occurrence of pairs of metabolites in metabolic reactions is termed herein "metabolite coupling." These metabolite pairs can be directly computed from the stoichiometric matrix, S. Metabolite coupling is derived from the matrix ŜŜT, whose off-diagonal elements indicate the number of reactions in which any two metabolites participate together, where Ŝ is the binary form of S. Results Metabolite coupling in the studied networks was found to be dominated by a relatively small group of highly interacting pairs of metabolites. As would be expected, metabolites with high individual metabolite connectivity also tended to be those with the highest metabolite coupling, as the most connected metabolites couple more often. For metabolite pairs that are not highly coupled, we show that the number of reactions a pair of metabolites shares across a metabolic network closely approximates a line on a log-log scale. We also show that the preferential coupling of two metabolites with each other is spread across the spectrum of metabolites and is not unique to the most connected metabolites. We provide a measure for determining which metabolite pairs couple more often than would be expected based on their individual connectivity in the network and show that these metabolites often derive their principal biological functions from existing in pairs. Thus, analysis of metabolite coupling provides information beyond that which is found from studying the individual connectivity of individual

Engineering Microbial Metabolite Dynamics and Heterogeneity.

Science.gov (United States)

Schmitz, Alexander C; Hartline, Christopher J; Zhang, Fuzhong

2017-10-01

As yields for biological chemical production in microorganisms approach their theoretical maximum, metabolic engineering requires new tools, and approaches for improvements beyond what traditional strategies can achieve. Engineering metabolite dynamics and metabolite heterogeneity is necessary to achieve further improvements in product titers, productivities, and yields. Metabolite dynamics, the ensemble change in metabolite concentration over time, arise from the need for microbes to adapt their metabolism in response to the extracellular environment and are important for controlling growth and productivity in industrial fermentations. Metabolite heterogeneity, the cell-to-cell variation in a metabolite concentration in an isoclonal population, has a significant impact on ensemble productivity. Recent advances in single cell analysis enable a more complete understanding of the processes driving metabolite heterogeneity and reveal metabolic engineering targets. The authors present an overview of the mechanistic origins of metabolite dynamics and heterogeneity, why they are important, their potential effects in chemical production processes, and tools and strategies for engineering metabolite dynamics and heterogeneity. The authors emphasize that the ability to control metabolite dynamics and heterogeneity will bring new avenues of engineering to increase productivity of microbial strains. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

Directory of Open Access Journals (Sweden)

Huzoor Akbar

Full Text Available Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation.We utilized the Mx-cre;Cdc42(lox/lox inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+ mice. Platelets isolated from Cdc42(-/-, as compared to Cdc42(+/+, mice exhibited (a diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b inhibition of filopodia formation on immobilized CRP or fibrinogen, (c inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/- mice compared with Cdc42(+/+ mice.Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

Identification of a tryptanthrin metabolite in rat liver microsomes by liquid chromatography/electrospray ionization-tandem mass spectrometry.

Science.gov (United States)

Lee, Sang Kyu; Kim, Ghee Hwan; Kim, Dong Hyeon; Kim, Dong Hyun; Jahng, Yurngdong; Jeong, Tae Cheon

2007-10-01

Tryptanthrin originally isolated from Isatis tinctoria L. has been characterized to have anti-inflammatory activities through the dual inhibition of cyclooxygenase-2 and 5-lipoxygenase mediated prostaglandin and leukotriene syntheses. To characterize phase I metabolite(s), tryptanthrin was incubated with rat liver microsomes in the presence of NADPH-generating system. One metabolite was identified by liquid chromatography/electrospray ionization-tandem mass spectrometry. M1 could be identified as a metabolite mono-hydroxylated on the aromatic ring of indole moiety from the MS(2) spectra of protonated tryptanthrin and M1. The structure of metabolite was confirmed as 8-hydroxytryptanthrin with a chemically synthesized authentic standard. The formation of M1 was NADPH-dependent and was inhibited by SKF-525A, a general CYP-inhibitor, indicating the cytochrome P450 (CYP)-mediated reaction. In addition, it was proposed that M1 might be formed by CYP 1A in rat liver microsomes from the experiments with enriched rat liver microsomes.

Effect of rosuvastatin dose-loading on serum sLox-1, hs-CRP, and postoperative prognosis in diabetic patients with acute coronary syndromes undergoing selected percutaneous coronary intervention (PCI).

Science.gov (United States)

Jiao, Yungen; Hu, Feng; Zhang, Zhengang; Gong, Kaizheng; Sun, Xiaoning; Li, Aihua; Liu, Naifeng

2015-01-01

To investigate the effect of rosuvastatin dose-loading on serum levels of lectin-like oxidized low-density lipoprotein receptor-1 (Lox-1) and high-sensitivity c-reactive protein (hs-CRP) and postoperative prognosis in patients with diabetes and non-ST segment elevation acute coronary syndromes (NSTEACS) undergoing selected percutaneous coronary intervention (PCI). A total of 72 patients with diabetes and NSTEACS were randomized to either the group treated with 20 mg rosuvastatin 12 hours prior to PCI with a second dose administered just before PCI (n = 33), or a control group treated with standard method according guideline (n = 39). Serum levels of sLox-1, hs-CRP, CK-MB, and cTnI were measured prior to PCI, and at 24 hours and 30 days after PCI. The 30-day incidence of major adverse cardiac events (MACE) was recorded in both groups. Compared to pre-PCI, serum levels of sLox-1 and hs-CRP of the two groups were increased at 24 hours after PCI (P PCI (P PCI, the levels of TC and LDL-C were not changed at 24 hours after PCI (P > 0.05) until 30 days after PCI (P PCI; the 30-day incidence of MACE occurred in 6.06% of patients in the loading-dose rosuvastatin-treated group and in 23.08% of patients in the control-treated group (P PCI, and also reduce the occurrence of MACE 30 days after PCI.

PCB 28 metabolites elimination kinetics in human plasma on a real case scenario: Study of hydroxylated polychlorinated biphenyl (OH-PCB) metabolites of PCB 28 in a highly exposed German Cohort.

Science.gov (United States)

Quinete, Natalia; Esser, André; Kraus, Thomas; Schettgen, Thomas

2017-07-05

Polychlorinated biphenyls (PCBs) are suspected of carcinogenic, neurotoxic and immunotoxic effects in animals and humans. Although background levels of PCBs have been slowly decreased after their ban, they are still among the most persistent and ubiquitous pollutants in the environment, remaining the subject of great concern. PCB 28 is a trichlorinated PCB found in high concentrations not only in human plasma but also in indoor air in Europe, yet little is known about its metabolic pathway and potential metabolites in humans. The present study aims to elucidate the kinetics of metabolite formation and elimination by analyzing four hydroxylated PCBs (OH-PCBs) in human plasma as potential metabolites of the PCB 28 congener. For this purpose, the study was conducted in plasma samples of highly PCB-exposed individuals (N=268), collected from 2010 to 2014 as a representation of a real case scenario with longitudinal data. OH-PCBs have been predicted, synthesized in the course of this study and further identified and quantitated in human plasma. This is the first time that previously unknown PCB 28 metabolites have been measured in human plasma and half-lives have been estimated for PCB metabolites, which could then provide further understanding in the toxicological consequences of exposure to PCBs in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of high pressure treatment on metabolite profile of marinated meat in soy sauce.

Science.gov (United States)

Yang, Yang; Ye, Yangfang; Wang, Ying; Sun, Yangying; Pan, Daodong; Cao, Jinxuan

2018-02-01

Marinated meat in soy sauce was produced using hind leg by washing, rubbing salt, marinating with soy sauce and spices, and air dry-ripening for 15d. The effect of high pressure (HP) (150 and 300MPa for 15min) on the metabolite profiles of products was characterized using 1 H NMR and multivariate data analysis. The results showed that the metabonome was dominated by 26 metabolites, including amino acids, sugars, organic acids, nucleic aides and their derivatives. PC1 and PC2 explained a total of 75.4 and 11.9% of variables, respectively. HP treatments increased most of the metabolites, especially PC1, glutamate, sugars, nucleotides, anserine, lactate and creatine compared to the control. The increase of metabolites under HP was not dependent on pressure level except for alanine, lactate, acetate, formate, fumarate, glucose and 5'-IMP. These findings demonstrated that HP treatment at 150MPa was economical to improve the taste of marinated meat in soy sauce. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolite Profiling of Red Sea Corals

KAUST Repository

Ortega, Jovhana Alejandra

2016-12-01

Looking at the metabolite profile of an organism provides insights into the metabolomic state of a cell and hence also into pathways employed. Little is known about the metabolites produced by corals and their algal symbionts. In particular, corals from the central Red Sea are understudied, but interesting study objects, as they live in one of the warmest and most saline environments and can provide clues as to the adjustment of corals to environmental change. In this study, we applied gas chromatography – mass spectrometry (GC–MS) metabolite profiling to analyze the metabolic profile of four coral species and their associated symbionts: Fungia granulosa, Acropora hemprichii, Porites lutea, and Pocillopora verrucosa. We identified and quantified 102 compounds among primary and secondary metabolites across all samples. F. granulosa and its symbiont showed a total of 59 metabolites which were similar to the 51 displayed by P. verrucosa. P. lutea and A. hemprichii both harbored 40 compounds in conjunction with their respective isolated algae. Comparing across species, 28 metabolites were exclusively present in algae, while 38 were exclusive to corals. A principal component and cluster analyses revealed that metabolite profiles clustered between corals and algae, but each species harbored a distinct catalog of metabolites. The major classes of compounds were carbohydrates and amino acids. Taken together, this study provides a first description of metabolites of Red Sea corals and their associated symbionts. As expected, the metabolites of coral hosts differ from their algal symbionts, but each host and algal species harbor a unique set of metabolites. This corroborates that host-symbiont species pairs display a fine-tuned complementary metabolism that provide insights into the specific nature of the symbiosis. Our analysis also revealed aquatic pollutants, which suggests that metabolite profiling might be used for monitoring pollution levels and assessing

Effect of phospholipid metabolites on model membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Shragin, A.S.; Vasilenko, I.A.; Selishcheva, A.A.; Shvets, V.I.

1985-01-01

/sup 31/P-NMR spectroscopy and formation of fluorescent complexes between Tb/sup 3 +/ and dipicolinic acid were used to monitor liposome fusion and the effects of phospholipases C and D on the process. Phospholipase C was found highly efficient in initiating liposomal fusion, regardless of the phospholipid composition of the bilayer membranes. However, phospholipase D promoted liposomal fusion only in cases in which the membranes contained high concentrations of phospholipids incapable of forming bilayer membranes, such as phosphatidylethanolamine and cardiolipin. The mechanism of action of both enzymes in promoting liposomal fusion was ascribed to the generation of a metastable state in the membranes as a result of enzymatic formation of lipophilic metabolites 1,2-diacylglycerol and phosphatidic acid. The perturbation, or fluidity, of the liposomal membranes favored fusion on contact. 21 references, 4 figures.

Secondary metabolites from marine microorganisms.

Science.gov (United States)

Kelecom, Alphonse

2002-03-01

After 40 years of intensive research, chemistry of marine natural products has become a mature field. Since 1995, there are signals of decreased interest in the search of new metabolites from traditional sources such as macroalgae and octocorals, and the number of annual reports on marine sponges stabilized. On the contrary, metabolites from microorganisms is a rapidly growing field, due, at least in part, to the suspicion that a number of metabolites obtained from algae and invertebrates may be produced by associated microorganisms. Studies are concerned with bacteria and fungi, isolated from seawater, sediments, algae, fish and mainly from marine invertebrates such as sponges, mollusks, tunicates, coelenterates and crustaceans. Although it is still to early to define tendencies, it may be stated that the metabolites from microorganisms are in most cases quite different from those produced by the invertebrate hosts. Nitrogenated metabolites predominate over acetate derivatives, and terpenes are uncommon. Among the latter, sesquiterpenes, diterpenes and carotenes have been isolated; among nitrogenated metabolites, amides, cyclic peptides and indole alkaloids predominate.

Secondary metabolites from marine microorganisms

Directory of Open Access Journals (Sweden)

KELECOM ALPHONSE

2002-01-01

Full Text Available After 40 years of intensive research, chemistry of marine natural products has become a mature field. Since 1995, there are signals of decreased interest in the search of new metabolites from traditional sources such as macroalgae and octocorals, and the number of annual reports on marine sponges stabilized. On the contrary, metabolites from microorganisms is a rapidly growing field, due, at least in part, to the suspicion that a number of metabolites obtained from algae and invertebrates may be produced by associated microorganisms. Studies are concerned with bacteria and fungi, isolated from seawater, sediments, algae, fish and mainly from marine invertebrates such as sponges, mollusks, tunicates, coelenterates and crustaceans. Although it is still to early to define tendencies, it may be stated that the metabolites from microorganisms are in most cases quite different from those produced by the invertebrate hosts. Nitrogenated metabolites predominate over acetate derivatives, and terpenes are uncommon. Among the latter, sesquiterpenes, diterpenes and carotenes have been isolated; among nitrogenated metabolites, amides, cyclic peptides and indole alkaloids predominate.

Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction

DEFF Research Database (Denmark)

González-Santamaría, José; Villalba, María; Busnadiego, Oscar

2016-01-01

arrhythmias, and sudden cardiac death. Cardiac fibrosis is characterized by extensive deposition of collagen and also by increased stiffness as a consequence of enhanced collagen cross-linking. Members of the lysyl oxidase (LOX) family of enzymes are responsible for the formation of collagen cross......-links. This study investigates the contribution of LOX family members to the heart response to MI. METHODS AND RESULTS: Experimental MI was induced in C57BL/6 mice by permanent ligation of the left anterior descending coronary artery. The expression of LOX isoforms (LOX and LOXL1-4) was strongly increased upon MI...... resulted in reduced ventricular dilatation and improved cardiac function. CONCLUSION: LOX family members contribute significantly to the detrimental effects of cardiac remodelling, highlighting LOX inhibition as a potential therapeutic strategy for post-infarction recovery....

Structural requirements for bioactivation of anticonvulsants to cytotoxic metabolites in vitro.

Science.gov (United States)

Riley, R J; Kitteringham, N R; Park, B K

1989-01-01

The formation of cytotoxic metabolites from the anticonvulsants phenytoin and carbamazepine was investigated in vitro using a hepatic microsomal enzyme system and human mononuclear leucocytes as target cells. Both drugs were metabolised to cytotoxic products. In order to assess the structural requirements for this bioactivation, a series of structurally related compounds was investigated. It was found that molecules which contain either an amide function or an aryl ring may undergo activation in vitro, but only the metabolism-dependent toxicity of the latter is potentiated by pre-treatment of the target cells with an epoxide hydrolase inhibitor. Taken collectively, these data are consistent with the concept that reactive epoxide metabolites of both phenytoin and carbamazepine may produce toxicity in individuals with an inherited deficiency in epoxide hydrolase. PMID:2590607

Metabolites of the phenylurea herbicides chlorotoluron, diuron, isoproturon and linuron produced by the soil fungus Mortierella sp

International Nuclear Information System (INIS)

Badawi, Nora; Ronhede, Stig; Olsson, Stefan; Kragelund, Birthe B.; Johnsen, Anders H.; Jacobsen, Ole Stig; Aamand, Jens

2009-01-01

Phenylurea herbicides are used worldwide, and often pollute surface- and groundwater in concentrations exceeding the limit value for drinking water (0.1 μg l -1 ). Bacteria degrade phenylurea herbicides by successive N-dealkylation to substituted aniline products. Little is known about the corresponding fungal pathways, however. We here report degradation of chlorotoluron, diuron, isoproturon and linuron by the soil fungus Mortierella sp. Gr4. Degradation was fastest with linuron and resulted in successively dealkylated metabolites and 3,4-dichloroaniline. A major new metabolite was detected that has not yet been fully identified. Thin layer chromatography and nuclear magnetic resonance spectroscopy indicate that it is a non-aromatic diol. Degradation of isoproturon, chlorotoluron and diuron involved successive N-demethylation and, in the case of isoproturon and chlorotoluron, additional hydroxylation. A new hydroxylated isoproturon metabolite was detected. The study thus shows that the fungal pathways differ from the bacterial pathways and yield new metabolites of possible environmental concern. - Fungal degradation of phenylurea herbicides results in the formation of hydroxylated metabolites and 3,4-dichloroaniline.

Metabolites of the phenylurea herbicides chlorotoluron, diuron, isoproturon and linuron produced by the soil fungus Mortierella sp

Energy Technology Data Exchange (ETDEWEB)

Badawi, Nora; Ronhede, Stig [Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Ostervoldgade 10, DK-1350 Copenhagen K (Denmark); Olsson, Stefan [Section of Genetics and Microbiology, Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C (Denmark); Kragelund, Birthe B. [Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen N (Denmark); Johnsen, Anders H. [Department of Clinical Biochemistry, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen O (Denmark); Jacobsen, Ole Stig [Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Ostervoldgade 10, DK-1350 Copenhagen K (Denmark); Aamand, Jens, E-mail: jeaa@geus.d [Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Ostervoldgade 10, DK-1350 Copenhagen K (Denmark)

2009-10-15

Phenylurea herbicides are used worldwide, and often pollute surface- and groundwater in concentrations exceeding the limit value for drinking water (0.1 mug l{sup -1}). Bacteria degrade phenylurea herbicides by successive N-dealkylation to substituted aniline products. Little is known about the corresponding fungal pathways, however. We here report degradation of chlorotoluron, diuron, isoproturon and linuron by the soil fungus Mortierella sp. Gr4. Degradation was fastest with linuron and resulted in successively dealkylated metabolites and 3,4-dichloroaniline. A major new metabolite was detected that has not yet been fully identified. Thin layer chromatography and nuclear magnetic resonance spectroscopy indicate that it is a non-aromatic diol. Degradation of isoproturon, chlorotoluron and diuron involved successive N-demethylation and, in the case of isoproturon and chlorotoluron, additional hydroxylation. A new hydroxylated isoproturon metabolite was detected. The study thus shows that the fungal pathways differ from the bacterial pathways and yield new metabolites of possible environmental concern. - Fungal degradation of phenylurea herbicides results in the formation of hydroxylated metabolites and 3,4-dichloroaniline.

Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages.

Science.gov (United States)

Shoeb, Mohammad; Ramana, Kota V

2012-01-01

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response. Copyright © 2011 Elsevier Inc. All rights reserved.

Tracing the fate of sulfamethoxazole and its metabolites in subsurface: conceptualization and modelling

Science.gov (United States)

Rodriguez-Escales, Paula; Sanchez-Vila, Xavier

2016-04-01

The degradation of low adsorptive SMX in subsurface porous media is spatially and temporally variable. It depends on various environmental factors such as in situ redox potential, availability of nutrients, local soil characteristics, and temperature. Its degradation is better under anoxic conditions and by co-metabolism processes. In this work, we first develop a conceptual model of degradation of SMX under different redox conditions (denitrification and iron reducing conditions), characterizing the metabolite formation in each condition, and second, we construct a mathematical model that allows reproducing different experiments of SMX degradation reported in the literature. The model was validated using the experimental data from Barbieri et al. (2012), Nödler et al. (2012) and Mohatt et al. (2011). The model reproduces the reversible degradation of SMX under the presence of nitrous acid as an intermediate product of denitrification (it is the conjugate acid of nitrite), as well as, the metabolite formation (4-nitro-SMX and desamino SMX). In those experiments degradation was mediated by the transient formation of a diazonium cation, which was considered responsible of the substitution of the amine radical by a nitro radical, forming the 4-nitro-SMX. On the other hand, both the diazonium compound and the methanol present in the experiment produced a deamination in the SMX, producing desamino-SMX. The formation of these metabolites was unstable and they were retransformed to SMX. Concerning the iron conditions, SMX was degraded due to the oxidation of iron (Fe2+), which was previously oxidized from goethite due to the degradation of a pool of labile organic carbon. As the oxidation of iron occurred on the goethite surface, the best model to reproduce the SMX reduction was a power law rate. Our work is an attempt to properly formulate the degradation process of an emerging compound considering the real degradation mechanisms, rather than using an upscaled black

Diclofenac toxicity in human intestine ex vivo is not related to the formation of intestinal metabolites

NARCIS (Netherlands)

Niu, Xiaoyu; de Graaf, Inge A. M.; Langelaar-Makkinje, Miriam; Horvatovich, Peter; Groothuis, Geny M. M.

The use of diclofenac (DCF), a nonsteroidal anti-inflammatory drug, is associated with a high prevalence of gastrointestinal side effects. In vivo studies in rodents suggested that reactive metabolites of DCF produced by the liver or the intestine might be responsible for this toxicity. In the

LOX-1 ligands containing apolipoprotein B and carotid intima-media thickness in middle-aged community-dwelling US Caucasian and Japanese men.

Science.gov (United States)

Okamura, Tomonori; Sekikawa, Akira; Sawamura, Tatsuya; Kadowaki, Takashi; Barinas-Mitchell, Emma; Mackey, Rachel H; Kadota, Aya; Evans, Rhobert W; Edmundowicz, Daniel; Higashiyama, Aya; Nakamura, Yasuyuki; Abbott, Robert D; Miura, Katsuyuki; Fujiyoshi, Akira; Fujita, Yoshiko; Murakami, Yoshitaka; Miyamatsu, Naomi; Kakino, Akemi; Maegawa, Hiroshi; Murata, Kiyoshi; Horie, Minoru; Mitsunami, Kenichi; Kashiwagi, Atsunori; Kuller, Lewis H; Ueshima, Hirotsugu

2013-07-01

The serum level of LOX-1 ligand containing ApoB (LAB) may reflect atherogenicity better than LDL cholesterol (LDLC), total LDL particles and usual measurement of oxidized LDL. The association between LAB and intima-media thickness (IMT) of carotid artery was investigated by ultrasound in US and Japan men. Participants were 297 US Caucasian and 310 Japanese men, aged 40-49 years without past history of cardiovascular disease. Serum LAB levels were measured by ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody. Serum LAB levels [median (interquartile range), μg/L] were 1321 (936, 1730) in US Caucasians and 940 (688, 1259) in Japanese. For Caucasian men, average IMT was higher in higher LAB quartile, which was 0.653, 0.667, 0.688, and 0.702 mm, respectively (p for trend = 0.02). Linear regression analysis showed serum LAB was significantly associated with IMT after adjustment for LDLC or total LDL particles in addition to other traditional or novel risk factors for atherosclerosis such as C-reactive protein. However, there was no significant relationship between LAB and IMT in Japanese men. Serum LAB, a new candidate biomarker for residual risk, was associated with an increased carotid IMT in US Caucasian men independently of various risk factors; however, ethnic difference should be clarified in the future. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Arachidonic acid in health and disease with focus on hypertension and diabetes mellitus: A review

Directory of Open Access Journals (Sweden)

Undurti N. Das

2018-05-01

Full Text Available Arachidonic acid (AA 20:4n-6 is an essential component of cell membranes and modulates cell membrane fluidity. AA is metabolized by cyclo-oxygenase (COX, lipoxygenase (LOX and cytochrome P450 enzymes to form several metabolites that have important biological actions. Of all the actions, role of AA in the regulation of blood pressure and its ability to prevent both type 1 and type 2 diabetes mellitus seems to be interesting. Studies showed that AA and its metabolites especially, lipoxin A4 (LXA4 and epoxyeicosatrienoic acids (EETs, potent anti-inflammatory metabolites, have a crucial role in the pathobiology of hypertension and diabetes mellitus. AA, LXA4 and EETs regulate smooth muscle function and proliferation, voltage gated ion channels, cell membrane fluidity, membrane receptors, G-coupled receptors, PPARs, free radical generation, nitric oxide formation, inflammation, and immune responses that, in turn, participate in the regulation blood pressure and pathogenesis of diabetes mellitus. In this review, role of AA and its metabolites LXA4 and EETs in the pathobiology of hypertension, pre-eclampsia and diabetes mellitus are discussed. Based on several lines of evidences, it is proposed that a combination of aspirin and AA could be of benefit in the prevention and management of hypertension, pre-eclampsia and diabetes mellitus. Keywords: Arachidonic acid, Lipoxin A4, Hypertension, Pre-eclampsia, Diabetes mellitus, Inflammation, Cytokines, Free radicals, Nitric oxide

Transportable hyperpolarized metabolites

Science.gov (United States)

Ji, Xiao; Bornet, Aurélien; Vuichoud, Basile; Milani, Jonas; Gajan, David; Rossini, Aaron J.; Emsley, Lyndon; Bodenhausen, Geoffrey; Jannin, Sami

2017-01-01

Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically <1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude. PMID:28072398

Microsomal metabolism of trenbolone acetate metabolites ...

Science.gov (United States)

Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

Detection of a reactive metabolite of misonidazole in human urine

International Nuclear Information System (INIS)

Varghese, A.J.; Whitmore, G.F.

1984-01-01

Chemical studies have indicated that, following reduction of misonidazole to the hydroxylamine derivative, reaction with guanosine leads to the formation of a 2-carbon addition product of guanosine. In this study, the formation of the guanosine product is used to detect the presence of a reactive metabolite of misonidazole in the urine of patients treated with misonidazole. Urine samples were incubated with [ 14 C]guanosine and the guanosine product was separated by HPLC analysis. The quantities of product vary as much as 10-fold from patient to patient and it is suggested that the assay be useful as a predictor of patients susceptible to the development of peripheral neuropathy or other effects of misonidazole

Is cardiovascular disease in patients with diabetes associated with serum levels of MMP-2, LOX, and the elastin degradation products ELM and ELM-2?

DEFF Research Database (Denmark)

Rørdam Preil, Simone; Faarvang Thorsen, Anne-Sofie; Christiansen, Anne Lindegaard

2017-01-01

BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression i......BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered m......RNA-expression in diabetic patients, namely matrix metalloproteinase 2 (MMP-2), lysyl oxidase (LOX) and elastin itself. In this study we investigate whether the serum concentrations of elastin-related proteins correlate to signs of CVD in patients with T2DM. METHODS: Blood samples from 302 type 2 diabetic patients were...... analysed for MMP-2, LOX, and the elastin degradation products ELM and ELM2. The results were investigated for correlations to signs of CVD in different vascular territories, as determined by myocardial perfusion scintigraphy, carotid artery thickness and ankle-brachial blood pressure index. RESULTS: T2DM...

Identifying diseases-related metabolites using random walk.

Science.gov (United States)

Hu, Yang; Zhao, Tianyi; Zhang, Ningyi; Zang, Tianyi; Zhang, Jun; Cheng, Liang

2018-04-11

Metabolites disrupted by abnormal state of human body are deemed as the effect of diseases. In comparison with the cause of diseases like genes, these markers are easier to be captured for the prevention and diagnosis of metabolic diseases. Currently, a large number of metabolic markers of diseases need to be explored, which drive us to do this work. The existing metabolite-disease associations were extracted from Human Metabolome Database (HMDB) using a text mining tool NCBO annotator as priori knowledge. Next we calculated the similarity of a pair-wise metabolites based on the similarity of disease sets of them. Then, all the similarities of metabolite pairs were utilized for constructing a weighted metabolite association network (WMAN). Subsequently, the network was utilized for predicting novel metabolic markers of diseases using random walk. Totally, 604 metabolites and 228 diseases were extracted from HMDB. From 604 metabolites, 453 metabolites are selected to construct the WMAN, where each metabolite is deemed as a node, and the similarity of two metabolites as the weight of the edge linking them. The performance of the network is validated using the leave one out method. As a result, the high area under the receiver operating characteristic curve (AUC) (0.7048) is achieved. The further case studies for identifying novel metabolites of diabetes mellitus were validated in the recent studies. In this paper, we presented a novel method for prioritizing metabolite-disease pairs. The superior performance validates its reliability for exploring novel metabolic markers of diseases.

Metabolite Biometrics for the Differentiation of Individuals.

Science.gov (United States)

Hair, Mindy E; Mathis, Adrianna I; Brunelle, Erica K; Halámková, Lenka; Halámek, Jan

2018-04-17

Sweat is a biological fluid present on the skin surface of every individual and is known to contain amino acids as well as other low molecular weight compounds. (1) Each individual is inherently different from one another based on certain factors including, but not limited to, his/her genetic makeup, environment, and lifestyle. As such, the biochemical composition of each person greatly differs. The concentrations of the biochemical content within an individual's sweat are largely controlled by metabolic processes within the body that fluctuate regularly based on attributes such as age, sex, and activity level. Therefore, the concentrations of these sweat components are person-specific and can be exploited, as presented here, to differentiate individuals based on trace amounts of sweat. For this concept, we analyzed three model compounds-lactate, urea, and glutamate. The average absorbance change from each compound in sweat was determined using three separate bioaffinity-based systems: lactate oxidase coupled with horseradish peroxidase (LOx-HRP), urease coupled with glutamate dehydrogenase (UR-GlDH), and glutamate dehydrogenase alone (GlDH). After optimization of a linear dependence for each assay to its respective analyte, analysis was performed on 50 mimicked sweat samples. Additionally, a collection and extraction method was developed and optimized by our group to evaluate authentic sweat samples from the skin surface of 25 individuals. A multivariate analysis of variance (MANOVA) test was performed to demonstrate that these three single-analyte enzymatic assays were effectively used to identify each person in both sample sets. This novel sweat analysis approach is capable of differentiating individuals, without the use of DNA, based on the collective responses from the chosen metabolic compounds in sweat. Applications for this newly developed, noninvasive analysis can include the field of forensic science in order to differentiate between individuals as well

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

Science.gov (United States)

Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

2015-03-01

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental

The Cremeomycin Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation.

Science.gov (United States)

Waldman, Abraham J; Pechersky, Yakov; Wang, Peng; Wang, Jennifer X; Balskus, Emily P

2015-10-12

Diazo groups are found in a range of natural products that possess potent biological activities. Despite longstanding interest in these metabolites, diazo group biosynthesis is not well understood, in part because of difficulties in identifying specific genes linked to diazo formation. Here we describe the discovery of the gene cluster that produces the o-diazoquinone natural product cremeomycin and its heterologous expression in Streptomyces lividans. We used stable isotope feeding experiments and in vitro characterization of biosynthetic enzymes to decipher the order of events in this pathway and establish that diazo construction involves late-stage N-N bond formation. This work represents the first successful production of a diazo-containing metabolite in a heterologous host, experimentally linking a set of genes with diazo formation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prioritizing Candidate Disease Metabolites Based on Global Functional Relationships between Metabolites in the Context of Metabolic Pathways

Science.gov (United States)

Yang, Haixiu; Xu, Yanjun; Han, Junwei; Li, Jing; Su, Fei; Zhang, Yunpeng; Zhang, Chunlong; Li, Dongguo; Li, Xia

2014-01-01

Identification of key metabolites for complex diseases is a challenging task in today's medicine and biology. A special disease is usually caused by the alteration of a series of functional related metabolites having a global influence on the metabolic network. Moreover, the metabolites in the same metabolic pathway are often associated with the same or similar disease. Based on these functional relationships between metabolites in the context of metabolic pathways, we here presented a pathway-based random walk method called PROFANCY for prioritization of candidate disease metabolites. Our strategy not only takes advantage of the global functional relationships between metabolites but also sufficiently exploits the functionally modular nature of metabolic networks. Our approach proved successful in prioritizing known metabolites for 71 diseases with an AUC value of 0.895. We also assessed the performance of PROFANCY on 16 disease classes and found that 4 classes achieved an AUC value over 0.95. To investigate the robustness of the PROFANCY, we repeated all the analyses in two metabolic networks and obtained similar results. Then we applied our approach to Alzheimer's disease (AD) and found that a top ranked candidate was potentially related to AD but had not been reported previously. Furthermore, our method was applicable to prioritize the metabolites from metabolomic profiles of prostate cancer. The PROFANCY could identify prostate cancer related-metabolites that are supported by literatures but not considered to be significantly differential by traditional differential analysis. We also developed a freely accessible web-based and R-based tool at http://bioinfo.hrbmu.edu.cn/PROFANCY. PMID:25153931

Pathway markers for pro-resolving lipid mediators in maternal and umbilical cord blood: A Secondary analysis of the Mothers, Omega-3, & Mental Health Study

Directory of Open Access Journals (Sweden)

Ellen L Mozurkewich

2016-09-01

Full Text Available The omega-3 fatty acids docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA are precursors to immune regulatory and specialized pro-resolving mediators (SPM of inflammation termed resolvins, maresins, and protections. Evidence for lipid mediator formation in vivo can be gained through evaluation of their 5-lipoxygenase (LOX and 15-LOX metabolic pathway precursors and downstream metabolites: We performed a secondary blood sample analysis from 60 participants in the Mothers, Omega-3, and Mental Health study to determine whether SPM and SPM precursors are augmented by dietary EPA- and DHA-rich fish oil supplementation compared to soy oil placebo. We also aimed to study whether SPM and their precursors differ in early and late pregnancy or between maternal and umbilical cord blood. We found that compared to placebo supplementation, EPA- and DHA- rich fish oil supplementation increased SPM precursor 17-HDHA concentrations in maternal and umbilical cord blood (P=0.02 We found that the D-series resolvin pathway marker 17-HDHA increased significantly between enrollment and late pregnancy (P=0.049. Levels of both 14-HDHA, a maresin pathway marker, and 17-HDHA were significantly greater in umbilical cord blood than in maternal blood (P<0.001, both.

Suppression of allene oxide synthase 3 in potato increases degree of arbuscular mycorrhizal fungal colonization.

Science.gov (United States)

Morcillo, Rafael Jorge León; Navarrete, María Isabel Tamayo; Bote, Juan Antonio Ocampo; Monguio, Salomé Prat; García-Garrido, José Manuel

2016-01-15

Arbuscular mycorrhizal (AM) is a mutually beneficial interaction among higher plants and soil fungi of the phylum Glomeromycota. Numerous studies have pointed that jasmonic acid plays an important role in the development of the intraradical fungus. This compound belongs to a group of biologically active compounds known as oxylipins which are derived from the oxidative metabolism of polyunsaturated fatty acids. Studies of the regulatory role played by oxylipins in AM colonization have generally focused on jasmonates, while few studies exist on the 9-LOX pathway of oxylipins during AM formation. Here, the cDNA of Allene oxide synthase 3 (AOS3), a key enzyme in the 9-LOX pathway, was used in the RNA interference (RNAi) system to transform potato plants in order to suppress its expression. Results show increases in AOS3 gene expression and 9-LOX products in roots of wild type potato mycorrhizal plants. The suppression of AOS3 gene expression increases the percentage of root with mycorrhizal colonization at early stages of AM formation. AOS3 RNA interference lead to an induction of LOXA and 13-LOX genes, a reduction in AOS3 derived 9-LOX oxylipin compounds and an increase in jasmonic acid content, suggesting compensation between 9 and 13-LOX pathways. The results in a whole support the hypothesis of a regulatory role for the 9-LOX oxylipin pathway during mycorrhization. Copyright © 2015 Elsevier GmbH. All rights reserved.

The use of stable isotopes and gas chromatography/mass spectrometry in the identification of steroid metabolites in the equine

International Nuclear Information System (INIS)

Houghton, E.; Dumasia, M.C.; Teale, P.; Smith, S.J.; Cox, J.; Marshall, D.; Gower, D.B.

1990-01-01

Stable isotope gas chromatography/mass spectrometry has been used successfully in the elucidation of structures of urinary steroid metabolites in the horse and in the identification of metabolites isolated from in vivo perfusion and in vitro incubation studies using equine tissue preparations. Deuterium-labeled steroids, testosterone, dehydroepiandrosterone, and 5-androstene-3 beta,17 beta-diol have been synthesized by base-catalyzed isotope exchange methods and the products characterized by gas chromatography/mass spectrometry. [16,16(-2)H2]Dehydroepiandrosterone (plus radiolabeled dehydroepiandrosterone) was perfused into a testicular artery of a pony stallion and was shown to be metabolized into 2H2-labeled testosterone, 4-androstenedione, isomers of 5-androstene-3,17-diol, 19-hydroxytestosterone, and 19-hydroxy-4-androstenedione. In further studies, equine testicular minces have been incubated with 2H2-labeled and radiolabeled dehydroepiandrosterone and 5-androstene-3 beta, 17 beta-diol. The metabolites, whose identity was confirmed by stable isotope gas chromatography/mass spectrometry, proved the interconversion of the two substrates, as well as formation of testosterone and 4-androstenedione. The aromatization of dehydroepiandrosterone was also confirmed, together with the formation of an isomer of 5(10)-estrene-3,17-diol from both substrates showing 19-demethylation without concomitant aromatization. In studies of the feto-placental unit, the allantochorion was shown to aromatize [2H5]testosterone to [2H4]estradiol, the loss of one 2H from the substrate being consistent with aromatization of the A ring. The formation of 6-hydroxyestradiol was also confirmed in this study. The same technique has been valuable in determining the structure of two metabolites of nandrolone isolated from horse urine

The sunburn response in human skin is characterized by sequential eicosanoid profiles that may mediate its early and late phases.

Science.gov (United States)

Rhodes, Lesley E; Gledhill, Karl; Masoodi, Mojgan; Haylett, Ann K; Brownrigg, Margaret; Thody, Anthony J; Tobin, Desmond J; Nicolaou, Anna

2009-11-01

Sunburn is a commonly occurring acute inflammatory process, with dermal vasodilatation and leukocyte infiltration as central features. Ultraviolet (UV) B-induced hydrolysis of membrane phospholipids releases polyunsaturated fatty acids, and their subsequent metabolism by cyclooxygenases (COXs) and lipoxygenases (LOXs) may produce potent eicosanoid mediators modulating different stages of the inflammation. Our objective was to identify candidate eicosanoids formed during the sunburn reaction in relation to its clinical and histological course. We exposed skin of healthy humans (n=32) to UVB and, for 72 h, examined expression of proinflammatory and anti-inflammatory eicosanoids using LC/ESI-MS/MS, and examined immunohistochemical expression of COX-2, 12-LOX, 15-LOX, and leukocyte markers, while quantifying clinical erythema. We show that vasodilatory prostaglandins (PGs) PGE(2), PGF(2alpha), and PGE(3) accompany the erythema in the first 24-48 h, associated with increased COX-2 expression at 24 h. Novel, potent leukocyte chemoattractants 11-, 12-, and 8-monohydroxy-eicosatetraenoic acid (HETE) are elevated from 4 to 72 h, in association with peak dermal neutrophil influx at 24 h, and increased dermal CD3(+) lymphocytes and 12- and 15-LOX expression from 24 to 72 h. Anti-inflammatory metabolite 15-HETE shows later expression, peaking at 72 h. Sunburn is characterized by overlapping sequential profiles of increases in COX products followed by LOX products that may regulate subsequent events and ultimately its resolution.

PCPF-M model for simulating the fate and transport of pesticides and their metabolites in rice paddy field.

Science.gov (United States)

Boulange, Julien; Malhat, Farag; Thuyet, Dang Quoc; Watanabe, Hirozumi

2017-12-01

The PCPF-1 model was improved for forecasting the fate and transport of metabolites in addition to parent compounds in rice paddies. In the new PCPF-M model, metabolites are generated from the dissipation of pesticide applied in rice paddies through hydrolysis, photolysis and biological degradations. The methodology to parameterize the model was illustrated using two scenarios for which uncertainty and sensitivity analyses were also conducted. In a batch degradation experiment, the hourly forecasted concentrations of fipronil and its metabolites in paddy water were very accurate. In a field-scale experiment, the hourly forecasted concentrations of fipronil in paddy water and paddy soil were accurate while the corresponding daily forecasted concentrations of metabolites were adequate. The major contributors to the variation of the forecasted metabolite concentrations in paddy water and paddy soil were the formation fractions of the metabolites. The influence of uncertainty included in input parameters on the forecasted metabolite concentration was high during the peak concentration of metabolite in paddy water. In contrast, in paddy soil, the metabolite concentrations forecasted several days after the initial pesticide application were sensitive to the uncertainty incorporated in the input parameters. The PCPF-M model simultaneously forecasts the concentrations of a parent pesticide and up to three metabolites. The model was validated using fipronil and two of its metabolites in paddy water and paddy soil. The model can be used in the early stage of the pesticide registration process and in risk assessment analysis for the evaluation of pesticide exposure. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Serotonergic neurotoxic metabolites of ecstasy identified in rat brain.

Science.gov (United States)

Jones, Douglas C; Duvauchelle, Christine; Ikegami, Aiko; Olsen, Christopher M; Lau, Serrine S; de la Torre, Rafael; Monks, Terrence J

2005-04-01

The selective serotonergic neurotoxicity of 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) depends on their systemic metabolism. We have recently shown that inhibition of brain endothelial cell gamma-glutamyl transpeptidase (gamma-GT) potentiates the neurotoxicity of both MDMA and MDA, indicating that metabolites that are substrates for this enzyme contribute to the neurotoxicity. Consistent with this view, glutathione (GSH) and N-acetylcysteine conjugates of alpha-methyl dopamine (alpha-MeDA) are selective neurotoxicants. However, neurotoxic metabolites of MDMA or MDA have yet to be identified in brain. Using in vivo microdialysis coupled to liquid chromatography-tandem mass spectroscopy and a high-performance liquid chromatography-coulometric electrode array system, we now show that GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA are present in the striatum of rats administered MDMA by subcutaneous injection. Moreover, inhibition of gamma-GT with acivicin increases the concentration of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA in brain dialysate, and there is a direct correlation between the concentrations of metabolites in dialysate and the extent of neurotoxicity, measured by decreases in serotonin (5-HT) and 5-hydroxyindole acetic (5-HIAA) levels. Importantly, the effects of acivicin are independent of MDMA-induced hyperthermia, since acivicin-mediated potentiation of MDMA neurotoxicity occurs in the context of acivicin-mediated decreases in body temperature. Finally, we have synthesized 5-(N-acetylcystein-S-yl)-N-methyl-alpha-MeDA and established that it is a relatively potent serotonergic neurotoxicant. Together, the data support the contention that MDMA-mediated serotonergic neurotoxicity is mediated by the systemic formation of GSH and N-acetylcysteine conjugates of N-methyl-alpha-MeDA (and alpha-MeDA). The mechanisms by which such metabolites access the brain and produce selective

Insights into the mechanisms of Promysalin, a secondary metabolite with genus-specific antibacterial activity against Pseudomonas

Science.gov (United States)

Promysalin, a secondary metabolite produced by Pseudomonas putida RW10S1, has antibacterial activity against a wide variety of Pseudomonas sp., including both human and plant pathogens. Promysalin induces swarming and biofilm formation in the producing species, and inhibits growth of susceptible sp...

Comparative Pharmacokinetics of Chlorpyrifos versus its Major Metabolites Following Oral Administration in the Rat

Energy Technology Data Exchange (ETDEWEB)

Busby-Hjerpe, Andrea L.; Campbell, James A.; Smith, Jordan N.; Lee, Sookwang; Poet, Torka S.; Barr, Dana; Timchalk, Charles

2010-01-31

Chlorpyrifos (CPF) is a commonly used diethylphosphorothionate organophosphorus (OP) insecticide. Diethylphosphate (DEP), diethylthiophosphate (DETP) and 3,5,6-trichloro-2-pyridinol (TCPy) are products of in vivo metabolism and environmental degradation of CPF and are routinely measured in urine as biomarkers of exposure. Hence, urinary biomonitoring of TCPy, DEP and DETP may be reflective of an individual’s contact with both the parent pesticide and exposure to these metabolites. In the current study, simultaneous dosing of 13C- or 2H- isotopically labeled CPF (13Clabeled CPF, 5 13C on the TCPy ring; or 2H-labeled CPF, diethyl-D10 (deuterium labeled) on the side chain) were exploited to directly compare the pharmacokinetics and metabolism of CPF with TCPy, and DETP. Individual metabolites were co-administered (oral gavage) with the parent compound at equal molar doses (14 μmol/kg; ~5mg/kg CPF). The key objective in the current study was to quantitatively evaluate the pharmacokinetics of the individual metabolites relative to their formation following a dose of CPF. Major differences in the pharmacokinetics between CPF and metabolites doses were observed within the first 3 h of exposure, due to the required metabolism of CPF to initially form TCPy and DETP. Nonetheless, once a substantial amount of CPF has been metabolized (≥ 3 h post-dosing) pharmacokinetics for both treatment groups and metabolites were very comparable. Urinary excretion rates for orally administered TCPy and DETP relative to 13C-CPF or 2H-CPF derived 13C-TCPy and 2H-DETP were consistent with blood pharmacokinetics, and the urinary clearance of metabolite dosed groups were comparable with the results for the 13C- and 2H-CPF groups. Since the pharmacokinetics of the individual metabolites were not modified by co-exposure to 3 CPF; it suggests that environmental exposure to low dose mixtures of pesticides and metabolites will not impact the pharmacokinetics of either.

Synthesis of Linezolid Metabolites PNU-142300 and PNU-142586 toward the Exploration of Metabolite-Related Events.

Science.gov (United States)

Hanaya, Kengo; Matsumoto, Kazuaki; Yokoyama, Yuta; Kizu, Junko; Shoji, Mitsuru; Sugai, Takeshi

2017-01-01

Linezolid (1) is an oxazolidinone antibiotic that is partially metabolized in vivo via ring cleavage of its morpholine moiety to mainly form two metabolites, PNU-142300 (2) and PNU-142586 (3). It is supposed that accumulation of 2 and 3 in patients with renal insufficiency may cause thrombocytopenia, one of the adverse effects of linezolid. However, the poor availability of 2 and 3 has hindered further investigation of the clinical significance of the accumulation of these metabolites. In this paper, we synthesized metabolites 2 and 3 via a common synthetic intermediate, 4; this will encourage further exploration of events related to these metabolites and lead to improved clinical use of linezolid.

Sequential metabolism of secondary alkyl amines to metabolic-intermediate complexes: opposing roles for the secondary hydroxylamine and primary amine metabolites of desipramine, (s)-fluoxetine, and N-desmethyldiltiazem.

Science.gov (United States)

Hanson, Kelsey L; VandenBrink, Brooke M; Babu, Kantipudi N; Allen, Kyle E; Nelson, Wendel L; Kunze, Kent L

2010-06-01

Three secondary amines desipramine (DES), (S)-fluoxetine [(S)-FLX], and N-desmethyldiltiazem (MA) undergo N-hydroxylation to the corresponding secondary hydroxylamines [N-hydroxydesipramine, (S)-N-hydroxyfluoxetine, and N-hydroxy-N-desmethyldiltiazem] by cytochromes P450 2C11, 2C19, and 3A4, respectively. The expected primary amine products, N-desmethyldesipramine, (S)-norfluoxetine, and N,N-didesmethyldiltiazem, are also observed. The formation of metabolic-intermediate (MI) complexes from these substrates and metabolites was examined. In each example, the initial rates of MI complex accumulation followed the order secondary hydroxylamine > secondary amine > primary amine, suggesting that the primary amine metabolites do not contribute to formation of MI complexes from these secondary amines. Furthermore, the primary amine metabolites, which accumulate in incubations of the secondary amines, inhibit MI complex formation. Mass balance studies provided estimates of the product ratios of N-dealkylation to N-hydroxylation. The ratios were 2.9 (DES-CYP2C11), 3.6 [(S)-FLX-CYP2C19], and 0.8 (MA-CYP3A4), indicating that secondary hydroxylamines are significant metabolites of the P450-mediated metabolism of secondary alkyl amines. Parallel studies with N-methyl-d(3)-desipramine and CYP2C11 demonstrated significant isotopically sensitive switching from N-demethylation to N-hydroxylation. These findings demonstrate that the major pathway to MI complex formation from these secondary amines arises from N-hydroxylation rather than N-dealkylation and that the primary amines are significant competitive inhibitors of MI complex formation.

Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance

Directory of Open Access Journals (Sweden)

Salme eTimmusk

2015-05-01

Full Text Available Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are nonribosomal peptide and polyketide derived metabolites (NRP/PK. Modular nonribosomal peptide synthetases catalyse main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 sfp-type phosphopantetheinyl transferase. The inactivation of the gene resulted in loss of NRP/PK production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. Its biofilm promotion is directly mediated by NRP/PK, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type.

Pesticide nonextractable residue formation in soil: insights from inverse modeling of degradation time series.

Science.gov (United States)

Loos, Martin; Krauss, Martin; Fenner, Kathrin

2012-09-18

Formation of soil nonextractable residues (NER) is central to the fate and persistence of pesticides. To investigate pools and extent of NER formation, an established inverse modeling approach for pesticide soil degradation time series was evaluated with a Monte Carlo Markov Chain (MCMC) sampling procedure. It was found that only half of 73 pesticide degradation time series from a homogeneous soil source allowed for well-behaved identification of kinetic parameters with a four-pool model containing a parent compound, a metabolite, a volatile, and a NER pool. A subsequent simulation indeed confirmed distinct parameter combinations of low identifiability. Taking the resulting uncertainties into account, several conclusions regarding NER formation and its impact on persistence assessment could nonetheless be drawn. First, rate constants for transformation of parent compounds to metabolites were correlated to those for transformation of parent compounds to NER, leading to degradation half-lives (DegT50) typically not being larger than disappearance half-lives (DT50) by more than a factor of 2. Second, estimated rate constants were used to evaluate NER formation over time. This showed that NER formation, particularly through the metabolite pool, may be grossly underestimated when using standard incubation periods. It further showed that amounts and uncertainties in (i) total NER, (ii) NER formed from the parent pool, and (iii) NER formed from the metabolite pool vary considerably among data sets at t→∞, with no clear dominance between (ii) and (iii). However, compounds containing aromatic amine moieties were found to form significantly more total NER when extrapolating to t→∞ than the other compounds studied. Overall, our study stresses the general need for assessing uncertainties, identifiability issues, and resulting biases when using inverse modeling of degradation time series for evaluating persistence and NER formation.

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Science.gov (United States)

Balazy, M

1991-12-15

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate

Identification of drug metabolites in human plasma or serum integrating metabolite prediction, LC-HRMS and untargeted data processing

NARCIS (Netherlands)

Jacobs, P.L.; Ridder, L.; Ruijken, M.; Rosing, H.; Jager, N.G.L.; Beijnen, J.H.; Bas, R.R.; Dongen, W.D. van

2013-01-01

Background: Comprehensive identification of human drug metabolites in first-in-man studies is crucial to avoid delays in later stages of drug development. We developed an efficient workflow for systematic identification of human metabolites in plasma or serum that combines metabolite prediction,

Steady-state metabolite concentrations reflect a balance between maximizing enzyme efficiency and minimizing total metabolite load.

Directory of Open Access Journals (Sweden)

Naama Tepper

Full Text Available Steady-state metabolite concentrations in a microorganism typically span several orders of magnitude. The underlying principles governing these concentrations remain poorly understood. Here, we hypothesize that observed variation can be explained in terms of a compromise between factors that favor minimizing metabolite pool sizes (e.g. limited solvent capacity and the need to effectively utilize existing enzymes. The latter requires adequate thermodynamic driving force in metabolic reactions so that forward flux substantially exceeds reverse flux. To test this hypothesis, we developed a method, metabolic tug-of-war (mTOW, which computes steady-state metabolite concentrations in microorganisms on a genome-scale. mTOW is shown to explain up to 55% of the observed variation in measured metabolite concentrations in E. coli and C. acetobutylicum across various growth media. Our approach, based strictly on first thermodynamic principles, is the first method that successfully predicts high-throughput metabolite concentration data in bacteria across conditions.

Functional metabolite assemblies—a review

Science.gov (United States)

Aizen, Ruth; Tao, Kai; Rencus-Lazar, Sigal; Gazit, Ehud

2018-05-01

Metabolites are essential for the normal operation of cells and fulfill various physiological functions. It was recently found that in several metabolic disorders, the associated metabolites could self-assemble to generate amyloid-like structures, similar to canonical protein amyloids that have a role in neurodegenerative disorders. Yet, assemblies with typical amyloid characteristics are also known to have physiological function. In addition, many non-natural proteins and peptides presenting amyloidal properties have been used for the fabrication of functional nanomaterials. Similarly, functional metabolite assemblies are also found in nature, demonstrating various physiological roles. A notable example is the structural color formed by guanine crystals or fluorescent crystals in feline eyes responsible for enhanced night vision. Moreover, some metabolites have been used for the in vitro fabrication of functional materials, such as glycine crystals presenting remarkable piezoelectric properties or indigo films used to assemble organic semi-conductive electronic devices. Therefore, we believe that the study of metabolite assemblies is not only important in order to understand their role in normal physiology and in pathology, but also paves a new route in exploring the fabrication of organic, bio-compatible materials.

Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control.

Science.gov (United States)

Balcells, Georgina; Matabosch, Xavier; Ventura, Rosa

2017-07-01

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

Science.gov (United States)

Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

2016-05-16

The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

Cellular Immune Reactions of the Sunn Pest, Eurygaster integriceps, to the Entomopathogenic Fungus, Beauveria bassiana and Its Secondary Metabolites

Science.gov (United States)

Zibaee, Arash; Bandani, Ali Reza; Talaei-Hassanlouei, Reza; Malagoli, Davide

2011-01-01

In this study, five morphological types of circulating hemocytes were recognized in the hemolymph of the adult sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), namely prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids. The effects of the secondary metabolites of the entomopathogenic fungus Beauveria bassiana on cellular immune defenses of Eurygaster integriceps were investigated. The results showed that the fungal secondary metabolites inhibited phagocytic activity of E. integriceps hemocytes and hampered nodule formation. A reduction of phenoloxidase activity was also observed. The data suggest that B. bassiana produce secondary metabolites that disable several immune mechanisms allowing the fungus to overcome and then kill its host. This characteristic makes B. bassiana a promising model for biological control of insect pests such as E. integriceps. PMID:22233481

Analysis of substrate degradation, metabolite formation and microbial community responses in sand bioreactors treating winery wastewater: a comparative study.

Science.gov (United States)

Welz, P J; Palmer, Z; Isaacs, S; Kirby, B; le Roes-Hill, M

2014-12-01

There is a global need for the implementation of more cost-effective green technologies for the treatment of effluent from wineries. However, systems reliant on microbial biodegradation may be adversely affected by the highly seasonal character of cellar waste. In this study, the biodegradation of two different formulations of winery effluent in sand bioreactors was compared. The degradation of organic substrates and formation of metabolites was monitored by physicochemical analyses of pore water and final effluent samples. Changes in the bacterial community structures were detected using molecular fingerprinting. In wastewater with an overall COD of 2027 mg/L, a formulation with a high concentration of acetate (800 mg COD/L) was more recalcitrant to degradation than a formulation with a high concentration of glucose (800 mg COD/L). Ethanol, glucose and phenolics were degraded preferentially in the deeper layers of the sand bioreactors (average Eh 25 mV) than in the superficial layers (average Eh 102 mV). The redox status also played a pivotal role on the bacterial community composition. The study yielded valuable insight that can be utilized in the design (configuration and operation) of full scale sand bioreactors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metabolite damage and repair in metabolic engineering design

Energy Technology Data Exchange (ETDEWEB)

Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.; Bruner, Steven D.; Hanson, Andrew D.

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

Zyflamend reduces LTB4 formation and prevents oral carcinogenesis in a 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced hamster cheek pouch model.

Science.gov (United States)

Yang, Peiying; Sun, Zheng; Chan, Diana; Cartwright, Carrie A; Vijjeswarapu, Mary; Ding, Jibin; Chen, Xiaoxin; Newman, Robert A

2008-11-01

Aberrant arachidonic acid metabolism, especially altered cyclooxygenase and 5-lipoxygenase (LOX) activities, has been associated with chronic inflammation as well as carcinogenesis in human oral cavity tissues. Here, we examined the effect of Zyflamend, a product containing 10 concentrated herbal extracts, on development of 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced inflammation and oral squamous cell carcinoma (SCC). A hamster cheek pouch model was used in which 0.5% DMBA was applied topically onto the left cheek pouch of male Syrian golden hamsters either three times per week for 3 weeks (short term) or 6 weeks (long term). Zyflamend was then applied topically at one of three different doses (25, 50 and 100 microl) onto the left cheek pouch three times for 1 week (short-term study) or chronically for 18 weeks. Zyflamend significantly reduced infiltration of inflammatory cells, incidence of hyperplasia and dysplastic lesions, bromodeoxyuridine-labeling index as well as number of SCC in a concentration-dependent manner. Application of Zyflamend (100 microl) reduced formation of leukotriene B(4) (LTB(4)) by 50% compared with DMBA-treated tissues. The reduction of LTB(4) was concentration dependent. The effect of Zyflamend on inhibition of LTB(4) formation was further confirmed with in vitro cell-based assay. Adding LTB(4) to RBL-1 cells, a rat leukemia cell line expressing high levels of 5-LOX and LTA(4) hydrolase, partially blocked antiproliferative effect of Zyflamend. This study demonstrates that Zyflamend inhibited LTB(4) formation and modulated adverse histopathological changes in the DMBA-induced hamster cheek pouch model. The study suggests that Zyflamend might prevent oral carcinogenesis at the post-initiation stage.

Urinary excretion of androgen metabolites, comparison with excretion of radioactive metabolites after injection of [4-14C]testosterone

International Nuclear Information System (INIS)

Deslypere, J.P.; Sayed, A.; Vermeulen, A.; Wiers, P.W.

1981-01-01

The influence of age on the metabolic pattern of [4- 14 C]testosterone was studied in 20 young and 8 elderly males and compared to the metabolic pattern of endogenous androgens; the latter was also studied in 16 young and 8 elderly women. In both young and elderly males, androsterone and aetiocholanolone glucuronide represent 65% of [4- 14 C]testosterone metabolites: together with their suephoconjugates as well as with 5α- and 5β-androstane-3α, 17β-diol they represent even more than 75% of total urinary metabolites. The 5α/5β ratio of metabolites of [4- 14 C]testosterone was significantly (P 14 C]testosterone metabolites was generally higher than the ratio of metabolites of endogenous androgens, suggesting that the transformation of T to ring A saturated metabolites occurs at least partially in another compartment than the transformation of DHEA to these metabolites. For both [4- 14 C]testosterone and endogenous androgen metabolites we observed a statistically significant reduction of the 5α/5β ratio with age, a general phenomenon in both males and females. This reduction concern also 11-OH-androst-4-ene-3.17-dione metabolism. Neither sex hormone levels, nor specific binding seems to determine this age dependent shift; neither is there convincing evidence for latent hypothyroisism or liver dysfunction in the elderly. An age associated primary decrease of the 5α-reductase activity seems the most likely explanation. (author)

Secondary metabolites in fungus-plant interactions

Science.gov (United States)

Pusztahelyi, Tünde; Holb, Imre J.; Pócsi, István

2015-01-01

Fungi and plants are rich sources of thousands of secondary metabolites. The genetically coded possibilities for secondary metabolite production, the stimuli of the production, and the special phytotoxins basically determine the microscopic fungi-host plant interactions and the pathogenic lifestyle of fungi. The review introduces plant secondary metabolites usually with antifungal effect as well as the importance of signaling molecules in induced systemic resistance and systemic acquired resistance processes. The review also concerns the mimicking of plant effector molecules like auxins, gibberellins and abscisic acid by fungal secondary metabolites that modulate plant growth or even can subvert the plant defense responses such as programmed cell death to gain nutrients for fungal growth and colonization. It also looks through the special secondary metabolite production and host selective toxins of some significant fungal pathogens and the plant response in form of phytoalexin production. New results coming from genome and transcriptional analyses in context of selected fungal pathogens and their hosts are also discussed. PMID:26300892

SPE-NMR metabolite sub-profiling of urine

NARCIS (Netherlands)

Jacobs, D.M.; Spiesser, L.; Garnier, M.; Roo, de N.; Dorsten, van F.; Hollebrands, B.; Velzen, van E.; Draijer, R.; Duynhoven, van J.P.M.

2012-01-01

NMR-based metabolite profiling of urine is a fast and reproducible method for detection of numerous metabolites with diverse chemical properties. However, signal overlap in the (1)H NMR profiles of human urine may hamper quantification and identification of metabolites. Therefore, a new method has

Metabolite damage and repair in metabolic engineering design.

Science.gov (United States)

Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

2017-11-01

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

Trophic transfer of pyrene metabolites between aquatic invertebrates

International Nuclear Information System (INIS)

Carrasco Navarro, V.; Leppänen, M.T.; Kukkonen, J.V.K.; Godoy Olmos, S.

2013-01-01

The trophic transfer of pyrene metabolites was studied using Gammarus setosus as a predator and the invertebrates Lumbriculus variegatus and Chironomus riparius as prey. The results obtained by liquid scintillation counting confirmed that the pyrene metabolites produced by the aquatic invertebrates L. variegatus and C. riparius were transferred to G. setosus through the diet. More detailed analyses by liquid chromatography discovered that two of the metabolites produced by C. riparius appeared in the chromatograms of G. setosus tissue extracts, proving their trophic transfer. These metabolites were not present in chromatograms of G. setosus exclusively exposed to pyrene. The present study supports the trophic transfer of PAH metabolites between benthic macroinvertebrates and common species of an arctic amphipod. As some PAH metabolites are more toxic than the parent compounds, the present study raises concerns about the consequences of their trophic transfer and the fate and effects of PAHs in natural environments. - Highlights: ► The trophic transfer of pyrene metabolites between invertebrates was evaluated. ► Biotransformation of pyrene by L. variegatus and C. riparius is different. ► Metabolites produced by L. variegatus and C. riparius are transferred to G. setosus. ► Specifically, two metabolites produced by C. riparius were transferred. - Some of the pyrene metabolites produced by the model invertebrates L. variegatus and C. riparius are transferred to G. setosus through the diet, proving their trophic transfer.

Does simvastatin stimulate bone formation in vivo?

Directory of Open Access Journals (Sweden)

Chorev Michael

2003-04-01

Full Text Available Abstract Background Statins, potent compounds that inhibit cholesterol synthesis in the liver have been reported to induce bone formation, both in tissue culture and in rats and mice. To re-examine potential anabolic effects of statins on bone formation, we compared the activity of simvastatin (SVS to the known anabolic effects of PTH in an established model of ovariectomized (OVX Swiss-Webster mice. Methods Mice were ovariectomized at 12 weeks of age (T0, remained untreated for 5 weeks to allow development of osteopenia (T5, followed by treatment for 8 weeks (T13. Whole, trabecular and cortical femoral bone was analyzed by micro-computed tomography (micro CT. Liquid chromatography/mass spectrometry (LC/MS was used to detect the presence of SVS and its active metabolite, simvastatin β-hydroxy acid (SVS-OH in the mouse serum. Results Trabecular BV/TV at T13 was 4.2 fold higher in animals treated with PTH (80 micro-g/kg/day compared to the OVX-vehicle treated group (p in vivo study. Conclusions While PTH demonstrated the expected anabolic effect on bone, SVS failed to stimulate bone formation, despite our verification by LC/MS of the active SVS-OH metabolite in mouse serum. While statins have clear effects on bone formation in vitro, the formulation of existing 'liver-targeted' statins requires further refinement for efficacy in vivo.

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

Science.gov (United States)

Lee, I. Russel; Chow, Eve W. L.; Morrow, Carl A.; Djordjevic, Julianne T.; Fraser, James A.

2011-01-01

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine—three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°–40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection. PMID:21441208

Anti-Biofilm and Antivirulence Activities of Metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa

Directory of Open Access Journals (Sweden)

Jinwei Zhou

2017-05-01

Full Text Available This study reported the efficacy of the metabolites of Plectosphaerella cucumerina, one phyllosphere fungus from Orychophragmus violaceus, against Pseudomonas aeruginosa quorum sensing (QS and QS-regulated biofilms. The minimum inhibitory concentration (MIC of the ethyl acetate (EtOAc extract from P. cucumerina against P. aeruginosa PAO1 was 1.25 mg mL−1. At sub-MIC concentrations, P. cucumerina extract (0.25–1 mg mL−1 not only inhibited biofilm formation but also disrupted preformed biofilms of P. aeruginosa PAO1 without affecting its growth. Fluorescence and scanning electron microscope (SEM showed architectural disruption of the biofilms when treated with P. cucumerina metabolites. Further investigation demonstrated that metabolites in P. cucumerina attenuated the QS-dependent virulence factors. LC-MS/MS spectra coupled with experimentally standard samples suggested that patulin and emodin might act as the principal components possessing anti-biofilm and antivirulence activities. This is the first report of (1 the isolation of P. cucumerina from the phyllosphere of O. violaceus and (2 anti-biofilm, antivirulence, and biofilm disruption activities of this fungus. Thus, this study provides fascinating new pathways for screening antipathogenic agents.

LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

Science.gov (United States)

Trettin, Arne; Jordan, Jens; Tsikas, Dimitrios

2014-09-01

Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS. Copyright © 2013 Elsevier B.V. All rights reserved.

Production of Secondary Metabolites in Extreme Environments: Food- and Airborne Wallemia spp. Produce Toxic Metabolites at Hypersaline Conditions

DEFF Research Database (Denmark)

Jančič, Sašo; Frisvad, Jens Christian; Kocev, Dragi

2016-01-01

the genome data analysis of W. mellicola (previously W. sebi sensu lato) and W. ichthyophaga revealed a low number of secondary metabolites clusters, a substantial number of secondary metabolites were detected at different conditions. Machine learning analysis of the obtained dataset showed that NaCl has...... of salt or sugar. In relation to food safety, the effect of high salt and sugar concentrations on the production of secondary metabolites by this toxigenic fungus was investigated. The secondary metabolite profiles of 30 strains of the listed species were examined using general growth media, known...... to support the production of secondary metabolites, supplemented with different concentrations of NaCl, glucose and MgCl2. In more than two hundred extracts approximately one hundred different compounds were detected using high-performance liquid chromatography-diode array detection (HPLC-DAD). Although...

Secondary metabolites from Ganoderma.

Science.gov (United States)

Baby, Sabulal; Johnson, Anil John; Govindan, Balaji

2015-06-01

Ganoderma is a genus of medicinal mushrooms. This review deals with secondary metabolites isolated from Ganoderma and their biological significance. Phytochemical studies over the last 40years led to the isolation of 431 secondary metabolites from various Ganoderma species. The major secondary compounds isolated are (a) C30 lanostanes (ganoderic acids), (b) C30 lanostanes (aldehydes, alcohols, esters, glycosides, lactones, ketones), (c) C27 lanostanes (lucidenic acids), (d) C27 lanostanes (alcohols, lactones, esters), (e) C24, C25 lanostanes (f) C30 pentacyclic triterpenes, (g) meroterpenoids, (h) farnesyl hydroquinones (meroterpenoids), (i) C15 sesquiterpenoids, (j) steroids, (k) alkaloids, (l) prenyl hydroquinone (m) benzofurans, (n) benzopyran-4-one derivatives and (o) benzenoid derivatives. Ganoderma lucidum is the species extensively studied for its secondary metabolites and biological activities. Ganoderma applanatum, Ganoderma colossum, Ganoderma sinense, Ganoderma cochlear, Ganoderma tsugae, Ganoderma amboinense, Ganoderma orbiforme, Ganoderma resinaceum, Ganoderma hainanense, Ganoderma concinna, Ganoderma pfeifferi, Ganoderma neo-japonicum, Ganoderma tropicum, Ganoderma australe, Ganoderma carnosum, Ganoderma fornicatum, Ganoderma lipsiense (synonym G. applanatum), Ganoderma mastoporum, Ganoderma theaecolum, Ganoderma boninense, Ganoderma capense and Ganoderma annulare are the other Ganoderma species subjected to phytochemical studies. Further phytochemical studies on Ganoderma could lead to the discovery of hitherto unknown biologically active secondary metabolites. Copyright © 2015 Elsevier Ltd. All rights reserved.

The eicosanoid response to high dose UVR exposure of individuals prone and resistant to sunburn.

Science.gov (United States)

Nicolaou, Anna; Masoodi, Mojgan; Gledhill, Karl; Haylett, Ann Katarina; Thody, Anthony John; Tobin, Desmond John; Rhodes, Lesley Elizabeth

2012-02-01

High personal UVR doses can be gained during leisure activities, causing intense self-resolving inflammation (sunburn) of unprotected skin. UVR activates release of membrane fatty acids and upregulates their metabolism by cyclooxygenases (COX) and lipoxygenases (LOX) to different eicosanoids. While COX-derived prostaglandin (PG)E(2) is a potent mediator of sunburn vasodilatation, LOX-derived 15-hydroxyeicosatetraenoic acid (HETE) and its lipoxin metabolites may contribute to sunburn limitation. We explored the relationships between expression of these lipid mediators and the clinical and histological outcomes, comparing responses of individuals prone and more resistant to sunburn. An acute UVR exposure of 12 SED (standard erythema dose) was applied to buttock skin of 32 white Caucasians (n = 16 phototype I/II, n = 16 phototype III/IV), and over the subsequent 72 h assessments were made of skin erythema, immunohistochemical expression of leukocyte markers, COX-2, 12-LOX, 15-LOX and nitric oxide synthase (NOS), and eicosanoid levels by LC/ESI-MS/MS. Evidence of a significant inflammatory response was seen earlier in phototype I/II with regard to expression of erythema (4 h, p sunburn, having greater influence in those prone to sunburn than those more resistant, given the same high UVR exposure conditions. This journal is © The Royal Society of Chemistry and Owner Societies 2012

Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

Science.gov (United States)

Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-06-01

Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolic analysis of the increased adventitious rooting mutant of Artemisia annua reveals a role for the plant monoterpene borneol in adventitious root formation.

Science.gov (United States)

Tian, Na; Liu, Shuoqian; Li, Juan; Xu, Wenwen; Yuan, Lin; Huang, Jianan; Liu, Zhonghua

2014-08-01

Adventitious root (AR) formation is a critical process for plant clonal propagation. The role of plant secondary metabolites in AR formation is still poorly understood. Chemical and physical mutagenesis in combination with somatic variation were performed on Artemisia annua in order to obtain a mutant with changes in adventitious rooting and composition of plant secondary metabolites. Metabolic and morphological analyses of the iar (increased adventitious rooting) mutant coupled with in vitro assays were used to elucidate the relationship between plant secondary metabolites and AR formation. The only detected differences between the iar mutant and wild-type were rooting capacity and borneol/camphor content. Consistent with this, treatment with borneol in vitro promoted adventitious rooting in wild-type. The enhanced rooting did not continue upon removal of borneol. The iar mutant displayed no significant differences in AR formation upon treatment with camphor. Together, our results suggest that borneol promotes adventitious rooting whereas camphor has no effect on AR formation. © 2013 Scandinavian Plant Physiology Society.

Cultivar-Specific Changes in Primary and Secondary Metabolites in Pak Choi (Brassica Rapa, Chinensis Group by Methyl Jasmonate

Directory of Open Access Journals (Sweden)

Moo Jung Kim

2017-05-01

Full Text Available Glucosinolates, their hydrolysis products and primary metabolites were analyzed in five pak choi cultivars to determine the effect of methyl jasmonate (MeJA on metabolite flux from primary metabolites to glucosinolates and their hydrolysis products. Among detected glucosinolates (total 14 glucosinolates; 9 aliphatic, 4 indole and 1 aromatic glucosinolates, indole glucosinolate concentrations (153–229% and their hydrolysis products increased with MeJA treatment. Changes in the total isothiocyanates by MeJA were associated with epithiospecifier protein activity estimated as nitrile formation. Goitrin, a goitrogenic compound, significantly decreased by MeJA treatment in all cultivars. Changes in glucosinolates, especially aliphatic, significantly differed among cultivars. Primary metabolites including amino acids, organic acids and sugars also changed with MeJA treatment in a cultivar-specific manner. A decreased sugar level suggests that they might be a carbon source for secondary metabolite biosynthesis in MeJA-treated pak choi. The result of the present study suggests that MeJA can be an effective agent to elevate indole glucosinolates and their hydrolysis products and to reduce a goitrogenic compound in pak choi. The total glucosinolate concentration was the highest in “Chinese cabbage” in the control group (32.5 µmol/g DW, but indole glucosinolates increased the greatest in “Asian” when treated with MeJA.

The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus

Directory of Open Access Journals (Sweden)

Pengxiang Chang

2018-02-01

Full Text Available Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI. Duck enteritis virus (DEV is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ and homology-directed repair (HDR. Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP, and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Secondary metabolites produced by marine streptomyces as antibiofilm and quorum-sensing inhibitor of uropathogen Proteus mirabilis.

Science.gov (United States)

Younis, Khansa Mohammed; Usup, Gires; Ahmad, Asmat

2016-03-01

Quorum-sensing regulates bacterial biofilm formation and virulence factors, thereby making it an interesting target for attenuating pathogens. In this study, we investigated anti-biofilm and anti-quorum-sensing compounds from secondary metabolites of halophiles marine streptomyces against urinary catheter biofilm forming Proteus mirabilis without effect on growth viability. A total of 40 actinomycetes were isolated from samples collected from different places in Iraq including marine sediments and soil samples. Fifteen isolates identified as streptomyces and their supernatant screened as anti-quorum-sensing by inhibiting quorum-sensing regulated prodigiosin biosynthesis of Serratia marcescens strain Smj-11 as a reporter strain. Isolate Sediment Lake Iraq (sdLi) showed potential anti-quorum-sensing activity. Out of 35 clinical isolates obtained from Urinary catheter used by patient at the Universiti Kebangsaan Malaysia Medical Center, 22 isolates were characterized and identified as Proteus mirabilis. Isolate Urinary Catheter B4 (UCB4) showed the highest biofilm formation with highest resistance to used antibiotic and was chosen for further studies. Ethyl acetate secondary metabolites extract was produced from sdLi isolate. First, we determined the Minimum Inhibitory Concentration (MIC) of sdLi crude extract against UCB4 isolate, and all further experiments used concentrations below the MIC. Tests of subinhibitory concentrations of sdLi crude extract showed good inhibition against UCB4 isolate biofilm formation on urinary catheter and cover glass using Scanning electron microscopy and light microscopy respectively. The influence of sub-MIC of sdLi crude extract was also found to attenuate the quorum sensing (QS)-dependent factors such as hemolysin activity, urease activity, pH value, and motility of UCB4 isolate. Evidence is presented that these nontoxic secondary metabolites may act as antagonists of bacterial quorum sensing by competing with quorum-sensing signals

Electronegative L5-LDL induces the production of G-CSF and GM-CSF in human macrophages through LOX-1 involving NF-κB and ERK2 activation.

Science.gov (United States)

Yang, Tzu-Ching; Chang, Po-Yuan; Kuo, Tzu-Ling; Lu, Shao-Chun

2017-12-01

Circulating levels of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) are associated with the severity of acute myocardial infarction (AMI). However, what causes increases in G-CSF and GM-CSF is unclear. In this study, we investigated whether L5-low-density lipoprotein (LDL), a mildly oxidized LDL from AMI, can induce G-CSF and GM-CSF production in human macrophages. L1-LDL and L5-LDL were isolated through anion-exchange chromatography from AMI plasma. Human macrophages derived from THP-1 and peripheral blood mononuclear cells were treated with L1-LDL, L5-LDL, or copper-oxidized LDL (Cu-oxLDL) and G-CSF and GM-CSF protein levels in the medium were determined. In addition, the effects of L5-LDL on G-CSF and GM-CSF production were tested in lectin-type oxidized LDL receptor-1 (LOX-1), CD36, extracellular signal-regulated kinase (ERK) 1, and ERK2 knockdown THP-1 macrophages. L5-LDL but not L1-LDL or Cu-oxLDL significantly induced production of G-CSF and GM-CSF in macrophages. In vitro oxidation of L1-LDL and L5-LDL altered their ability to induce G-CSF and GM-CSF, suggesting that the degree of oxidation is critical for the effects. Knockdown and antibody neutralization experiments suggested that the effects were caused by LOX-1. In addition, nuclear factor (NF)-κB and ERK1/2 inhibition resulted in marked reductions of L5-LDL-induced G-CSF and GM-CSF production. Moreover, knockdown of ERK2, but not ERK1, hindered L5-LDL-induced G-CSF and GM-CSF production. The results indicate that L5-LDL, a naturally occurring mild oxidized LDL, induced G-CSF and GM-CSF production in human macrophages through LOX-1, ERK2, and NF-κB dependent pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Yeast synthetic biology for high-value metabolites.

Science.gov (United States)

Dai, Zhubo; Liu, Yi; Guo, Juan; Huang, Luqi; Zhang, Xueli

2015-02-01

Traditionally, high-value metabolites have been produced through direct extraction from natural biological sources which are inefficient, given the low abundance of these compounds. On the other hand, these high-value metabolites are usually difficult to be synthesized chemically, due to their complex structures. In the last few years, the discovery of genes involved in the synthetic pathways of these metabolites, combined with advances in synthetic biology tools, has allowed the construction of increasing numbers of yeast cell factories for production of these metabolites from renewable biomass. This review summarizes recent advances in synthetic biology in terms of the use of yeasts as microbial hosts for the identification of the pathways involved in the synthesis, as well as for the production of high-value metabolites. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Novel urinary metabolite signature for diagnosing postpartum depression

Directory of Open Access Journals (Sweden)

Lin L

2017-05-01

Full Text Available Lin Lin, Xiao-mei Chen, Rong-hua Liu Department of Obstetrics and Gynecology, Linyi People’s Hospital, Shandong, People’s Republic of China Background: Postpartum depression (PPD could affect ~10% of women and impair the quality of mother–infant interactions. Currently, there are no objective methods to diagnose PPD. Therefore, this study was conducted to identify potential biomarkers for diagnosing PPD.Materials and methods: Morning urine samples of PPD subjects, postpartum women without depression (PPWD and healthy controls (HCs were collected. The gas chromatography-mass spectroscopy (GC-MS-based urinary metabolomic approach was performed to characterize the urinary metabolic profiling. The orthogonal partial least-squares-discriminant analysis (OPLS-DA was used to identify the differential metabolites. The logistic regression analysis and Bayesian information criterion rule were further used to identify the potential biomarker panel. The receiver operating characteristic curve analysis was conducted to evaluate the diagnostic performance of the identified potential biomarker panel.Results: Totally, 73 PPD subjects, 73 PPWD and 74 HCs were included, and 68 metabolites were identified using GC-MS. The OPLS-DA model showed that there were 22 differential metabolites (14 upregulated and 8 downregulated responsible for separating PPD subjects from HCs and PPWD. Meanwhile, a panel of five potential biomarkers – formate, succinate, 1-methylhistidine, a-glucose and dimethylamine – was identified. This panel could effectively distinguish PPD subjects from HCs and PPWD with an area under the curve (AUC curve of 0.948 in the training set and 0.944 in the testing set.Conclusion: These results demonstrated that the potential biomarker panel could aid in the future development of an objective diagnostic method for PPD. Keywords: postpartum depression, gas chromatography-mass spectroscopy, biomarker, metabolomics

Identification of metabolites of the tryptase inhibitor CRA-9249: observation of a metabolite derived from an unexpected hydroxylation pathway.

Science.gov (United States)

Yu, Walter; Dener, Jeffrey M; Dickman, Daniel A; Grothaus, Paul; Ling, Yun; Liu, Liang; Havel, Chris; Malesky, Kimberly; Mahajan, Tania; O'Brian, Colin; Shelton, Emma J; Sperandio, David; Tong, Zhiwei; Yee, Robert; Mordenti, Joyce J

2006-08-01

The metabolites of the tryptase inhibitor CRA-9249 were identified after exposure to liver microsomes. CRA-9249 was found to be degraded rapidly in liver microsomes from rabbit, dog, cynomolgus monkey, and human, and less rapidly in microsomes from rat. The key metabolites included cleavage of an aryl ether, in addition to an unexpected hydroxylation of the amide side chain adjacent to the amide nitrogen. The chemical structures of both metabolites were confirmed by synthesis and comparison to material isolated from the liver microsomes. Several suspected hydroxylated metabolites were also synthesized and analyzed as part of the structure identification process.

Characterization of midazolam metabolism in locusts: The role of CYP3A4-like enzyme in the formation of 1'-OH and 4-OH midazolam

DEFF Research Database (Denmark)

Olsen, Line Rørbæk; Gabel-Jensen, Charlotte; Wubshet, Sileshi Gizachew

2016-01-01

) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase...

The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production.

Science.gov (United States)

Akgul, Yucel; Derk, Raymond C; Meighan, Terence; Rao, K Murali Krishna; Murono, Eisuke P

2011-07-01

The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum. Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors. Published by Elsevier Inc.

Hydrophobicity and charge shape cellular metabolite concentrations.

Directory of Open Access Journals (Sweden)

Arren Bar-Even

2011-10-01

Full Text Available What governs the concentrations of metabolites within living cells? Beyond specific metabolic and enzymatic considerations, are there global trends that affect their values? We hypothesize that the physico-chemical properties of metabolites considerably affect their in-vivo concentrations. The recently achieved experimental capability to measure the concentrations of many metabolites simultaneously has made the testing of this hypothesis possible. Here, we analyze such recently available data sets of metabolite concentrations within E. coli, S. cerevisiae, B. subtilis and human. Overall, these data sets encompass more than twenty conditions, each containing dozens (28-108 of simultaneously measured metabolites. We test for correlations with various physico-chemical properties and find that the number of charged atoms, non-polar surface area, lipophilicity and solubility consistently correlate with concentration. In most data sets, a change in one of these properties elicits a ~100 fold increase in metabolite concentrations. We find that the non-polar surface area and number of charged atoms account for almost half of the variation in concentrations in the most reliable and comprehensive data set. Analyzing specific groups of metabolites, such as amino-acids or phosphorylated nucleotides, reveals even a higher dependence of concentration on hydrophobicity. We suggest that these findings can be explained by evolutionary constraints imposed on metabolite concentrations and discuss possible selective pressures that can account for them. These include the reduction of solute leakage through the lipid membrane, avoidance of deleterious aggregates and reduction of non-specific hydrophobic binding. By highlighting the global constraints imposed on metabolic pathways, future research could shed light onto aspects of biochemical evolution and the chemical constraints that bound metabolic engineering efforts.

New metabolites of hongdenafil, homosildenafil and hydroxyhomosildenafil.

Science.gov (United States)

Yeo, Miseon; Park, Yujin; Lee, Heesang; Choe, Sanggil; Baek, Seung-Hoon; Kim, Hye Kyung; Pyo, Jae Sung

2018-02-05

Recently, illegal sildenafil analogues have emerged, causing serious social issues. In spite of the importance of sildenafil analogues, their metabolic profiles or clinical effects have not been reported yet. In this study, new metabolites of illegal sildenafil analogues such as hongdenafil, homosildenafil, and hydroxyhomosildenafil were determined using liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS) and tandem mass spectrometry (LC-Q-TOF-MS/MS). To prepare metabolic samples, in vitro and in vivo studies were performed. For in vivo metabolites analysis, urine and feces samples of rats treated with sildenafil analogues were analyzed. For in vitro metabolites analysis, human liver microsomes incubated with sildenafil analogues were extracted and analyzed. All metabolites were characterized by LC-Q-TOF-MS and LC-Q-TOF-MS/MS. As a result, five, six, and seven metabolites were determined in hongdenafil, homosildenafil, and hydroxyhomosildenafil treated samples, respectively. These results could be applied to forensic science and other analytical fields. Moreover, these newly identified metabolites could be used as fundamental data to determine the side effect and toxicity of illegal sildenafil analogues. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

International Nuclear Information System (INIS)

Eto, Hideyuki; Miyata, Masaaki; Kume, Noriaki; Minami, Manabu; Itabe, Hiroyuki; Orihara, Koji; Hamasaki, Shuichi; Biro, Sadatoshi; Otsuji, Yutaka; Kita, Toru; Tei, Chuwa

2006-01-01

Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 μg/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury

Familial Resemblance for Serum Metabolite Concentrations

NARCIS (Netherlands)

Draisma, H.H.M.; Beekman, M.; Pool, R.; van Ommen, G.J.B; Vaarhorst, A.A.M.; de Craen, A.J.; Willemsen, G.; Slagboom, P.E.; Boomsma, D.I.

2013-01-01

Metabolomics is the comprehensive study of metabolites, which are the substrates, intermediate, and end products of cellular metabolism. The heritability of the concentrations of circulating metabolites bears relevance for evaluating their suitability as biomarkers for disease. We report aspects of

Metabolite Depletion Affects Flux Profiling of Cell Lines

DEFF Research Database (Denmark)

Nilsson, A.; Haanstra, J. R.; Teusink, B.

2018-01-01

Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation.......Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation....

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes

Science.gov (United States)

2012-01-01

Background Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. Results Fifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only. Conclusions Mycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites. PMID:22852578

Isolation and screening of proangiogenic and antiangiogenic metabolites producing rare actinobacteria from soil.

Science.gov (United States)

Azarakhsh, Y; Mohammadipanah, F; Nassiri, S M; Siavashi, V; Hamedi, J

2017-06-01

Angiogenesis is a physiological process that has important impacts on the pathology and healing of various diseases, and its induction or inhibition by bioactive actinobacterial metabolites can help the treatment of some diseases. In this study, the effects of actinobacterial extract in the process of angiogenesis have been explored. In this research, proangiogenic and antiangiogenic metabolites producing actinobacteria were isolated from soil samples and their fermentation broth were extracted and after evaluation of their toxicity by MTT assay, antiangiogenic and proangiogenic activities were screened against human umbilical vein endothelial cells (HUVECs) by in vitro tube formation and migration assay. Isolated strains were identified through molecular techniques. The results showed that Nocardiopsis arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts had a high potential of anti-angiogenic activity on HUVECs. For the first time proangiogenic potency of a rare actinobacterium, Kribbella sp. UTMC 522, was reported, and N. arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts inhibits the proliferation, migration and angiogenesis activity of HUVECs with reasonable potency. Metabolites of the introduced rare actinobacteria are potent proangiogenic and angiogenic inhibitors. Identification of angiogenic-antiangiogenic mechanisms and purification of the extracts would be useful in therapeutic angiogenesis. © 2017 The Society for Applied Microbiology.

Dissipation kinetics of asparagine in soil measured by compound-specific analysis with metabolite tracking

DEFF Research Database (Denmark)

Czaban, Weronika; Rasmussen, Jim; Nicolaisen, Mogens

2016-01-01

labeled glutamic acid were detected in soil. This highlights the fast turnover of amino acid in soil and that the estimation of concentration of the formed compounds is important when evaluating plant available organic N. Efficiency of the compound-specific analysis showed to be a powerful technique......Estimating the potential for direct plant acquisition of organic N, in particular amino acids, requires assessment of their turnover times in soil. It is well known from 14C studies that mineralization of amino acids occurs within hours, but mineralization to 14CO2 does not indicate the rate...... of disappearance of the intact amino acid or the possible formation of metabolites during amino acid dissipation. We here used compound-specific isotope analysis with metabolite tracking to investigate the dissipation rate of universally labeled intact 13C15N-asparagine at two concentrations and the subsequent...

Simvastatin (SV) metabolites in mouse tissues

International Nuclear Information System (INIS)

Duncan, C.A.; Vickers, S.

1990-01-01

SV, a semisynthetic analog of lovastatin, is hydrolyzed in vivo to its hydroxy acid (SVA), a potent inhibitor of HMG CoA reductase (HR). Thus SV lowers plasma cholesterol. SV is a substrate for mixed function oxidases whereas SVA undergoes lactonization and β-oxidation. Male CD-1 mice were dosed orally with a combination of ( 14 C)SV and ( 3 H)SVA at 25 mg/kg of each, bled and killed at 0.5, 2 and 4 hours. Labeled SV, SVA, 6'exomethylene SV (I), 6'CH 2 OH-SV (II), 6'COOH-SV (III) and a β-oxidized metabolite (IV) were assayed in liver, bile, kidneys, testes and plasma by RIDA. Levels of potential and active HR inhibitors in liver were 10 to 40 fold higher than in other tissues. II and III, in which the configuration at 6' is inverted, may be 2 metabolites of I. Metabolites I-III are inhibitors of HR in their hydroxy acid forms. Qualitatively ( 14 C)SV and ( 3 H)SVA were metabolized similarly (consistent with their proposed interconversion). However 3 H-SVA, I-III (including hydroxy acid forms) achieved higher concentrations than corresponding 14 C compounds (except in gall bladder bile). Major radioactive metabolites in liver were II-IV (including hydroxy acid forms). These metabolites have also been reported in rat tissues. In bile a large fraction of either label was unidentified polar metabolites. The presence of IV indicated that mice (like rats) are not good models for SV metabolism in man

Drug repositioning for enzyme modulator based on human metabolite-likeness.

Science.gov (United States)

Lee, Yoon Hyeok; Choi, Hojae; Park, Seongyong; Lee, Boah; Yi, Gwan-Su

2017-05-31

Recently, the metabolite-likeness of the drug space has emerged and has opened a new possibility for exploring human metabolite-like candidates in drug discovery. However, the applicability of metabolite-likeness in drug discovery has been largely unexplored. Moreover, there are no reports on its applications for the repositioning of drugs to possible enzyme modulators, although enzyme-drug relations could be directly inferred from the similarity relationships between enzyme's metabolites and drugs. We constructed a drug-metabolite structural similarity matrix, which contains 1,861 FDA-approved drugs and 1,110 human intermediary metabolites scored with the Tanimoto similarity. To verify the metabolite-likeness measure for drug repositioning, we analyzed 17 known antimetabolite drugs that resemble the innate metabolites of their eleven target enzymes as the gold standard positives. Highly scored drugs were selected as possible modulators of enzymes for their corresponding metabolites. Then, we assessed the performance of metabolite-likeness with a receiver operating characteristic analysis and compared it with other drug-target prediction methods. We set the similarity threshold for drug repositioning candidates of new enzyme modulators based on maximization of the Youden's index. We also carried out literature surveys for supporting the drug repositioning results based on the metabolite-likeness. In this paper, we applied metabolite-likeness to repurpose FDA-approved drugs to disease-associated enzyme modulators that resemble human innate metabolites. All antimetabolite drugs were mapped with their known 11 target enzymes with statistically significant similarity values to the corresponding metabolites. The comparison with other drug-target prediction methods showed the higher performance of metabolite-likeness for predicting enzyme modulators. After that, the drugs scored higher than similarity score of 0.654 were selected as possible modulators of enzymes for

Hydroquinone, a benzene metabolite, induces Hog1-dependent stress response signaling and causes aneuploidy in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Shiga, Takeki; Suzuki, Hiroyuki; Yamamoto, Hiroaki; Yamamoto, Kazuo; Yamamoto, Ayumi

2010-01-01

Previously, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test-negative carcinogen o-phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae by arresting the cell cycle at the G2/M transition as a result of the activation of the Hog1 (p38 MAPK homolog)-Swe1 (Wee1 homolog) pathway. In this experiment, we examined the aneuploidy forming effects of hydroquinone, a benzene metabolite, since both phenyl hydroquinone and hydroquinone are Ames-test negative carcinogens and share similar molecular structures. As was seen in phenyl hydroquinone, hydroquinone induced aneuploidy in yeast by delaying the cell cycle at the G2/M transition. Deficiencies in SWE1 and HOG1 abolished the hydroquinone-induced delay at the G2/M transition and aneuploidy formation. Furthermore, Hog1 was phosphorylated by hydroquinone, which may stabilize Swe1. These data indicate that the hydroquinone-induced G2/M transition checkpoint, which is activated by the Hog1-Swe1 pathway, plays a role in the formation of aneuploidy. (author)

Metabolites of cannabidiol identified in human urine.

Science.gov (United States)

Harvey, D J; Mechoulam, R

1990-03-01

1. Urine from a dystonic patient treated with cannabidiol (CBD) was examined by g.l.c.-mass spectrometry for CBD metabolites. Metabolites were identified as their trimethylsilyl (TMS), [2H9]TMS, and methyl ester/TMS derivatives and as the TMS derivatives of the product of lithium aluminium deuteride reduction. 2. Thirty-three metabolites were identified in addition to unmetabolized CBD, and a further four metabolites were partially characterized. 3. The major metabolic route was hydroxylation and oxidation at C-7 followed by further hydroxylation in the pentyl and propenyl groups to give 1"-, 2"-, 3"-, 4"- and 10-hydroxy derivatives of CBD-7-oic acid. Other metabolites, mainly acids, were formed by beta-oxidation and related biotransformations from the pentyl side-chain and these were also hydroxylated at C-6 or C-7. The major oxidized metabolite was CBD-7-oic acid containing a hydroxyethyl side-chain. 4. Two 8,9-dihydroxy compounds, presumably derived from the corresponding epoxide were identified. 5. Also present were several cyclized cannabinoids including delta-6- and delta-1-tetrahydrocannabinol and cannabinol. 6. This is the first metabolic study of CBD in humans; most observed metabolic routes were typical of those found for CBD and related cannabinoids in other species.

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

Energy Technology Data Exchange (ETDEWEB)

Hsieh, Y.-J. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Chen, Fu-Du [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Institute of Radiological Sciences, Central Taiwan University of Science and Technology, Taiwan (China); Wang, F.H. [National Yang-Ming University Medical School, Taiwan (China); Ke, C.C. [National PET/Cyclotron Center, Taipei Veterans General Hospital, Taiwan (China); Wang, H.-E. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Liu, R.-S. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China) and National Yang-Ming University Medical School, Taiwan (China) and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taiwan (China)]. E-mail: maimai5010@yahoo.com.tw

2007-02-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

International Nuclear Information System (INIS)

Hsieh, Y.-J.; Chen, Fu-Du; Wang, F.H.; Ke, C.C.; Wang, H.-E.; Liu, R.-S.

2007-01-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC

The effect of dose, dose rate, route of administration, and species on tissue and blood levels of benzene metabolites

International Nuclear Information System (INIS)

Henderson, R.F.; Sabourin, P.J.; Bechtold, W.E.; Griffith, W.C.; Medinsky, M.A.; Birnbaum, L.S.; Lucier, G.W.

1989-01-01

Studies were completed in F344/N rats and B6C3F 1 mice to determine the effect of dose, dose rate, route of administration, and rodent species on formation of total and individual benzene metabolites. Oral doses of 50 mg/kg or higher saturated the capacity for benzene metabolism in both rats and mice, resulting in an increased proportion of the administered dose being exhaled as benzene. The saturating air concentration for benzene metabolism during 6-hr exposures was between 130 and 900 ppm. At the highest exposure concentration, rats exhaled approximately half of the internal dose retained at the end of the 6-hr exposure as benzene; mice exhaled only 15% as benzene. Mice were able to convert more of the inhaled benzene to metabolites than were rats. In addition, mice metabolized more of the benzene by pathways leading to the putative toxic metabolites, benzoquinone and muconaldehyde, than did rats. In both rats and mice, the effect of increasing dose, administered orally or by inhalation, was to increase the proportion of the total metabolites that were the products of detoxification pathways relative to the products of pathways leading to putative toxic metabolites. This indicates low-affinity, high-capacity pathways for detoxification and high-affinity, low-capacity pathways leading to putative toxic metabolites. If the results of rodent studied performed at high doses were used to assess the health risk at low-dose exposures to benzene, the toxicity of benzene would be underestimated

Reactions of the melatonin metabolite N(1)-acetyl-5-methoxykynuramine with carbamoyl phosphate and related compounds.

Science.gov (United States)

Kuesel, Jana T; Hardeland, Rüdiger; Pfoertner, Henrike; Aeckerle, Nelia

2010-01-01

N-[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.

Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry.

Science.gov (United States)

Vikingsson, Svante; Josefsson, Martin; Gréen, Henrik

2015-01-01

The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of hepatic metabolites of two highly carcinogenic polycyclic aza-aromatic compounds, 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine.

Science.gov (United States)

Ye, Y; Duke, C C; Holder, G M

1995-03-01

The hepatic microsomal metabolites of the highly carcinogenic dimethylbenzacridines, 7,9-dimethylbenz[c]acridine (7,9-DMBAC), and 7,10-dimethylbenz[c]acridine (7,10-DMBAC) were obtained with preparations from 3-methylcholanthrene-pretreated rats. Metabolites were separated by reversed-phase HPLC and characterized using UV spectral data and chemical ionization-mass spectrometry after trimethylsilylation and GC. Comparisons with products formed in the presence of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide and with those formed from the three synthetic alcohol derivatives of each parent compound, aided the assignment of firm or tentative structures to 16 products from 7,9-DMBAC found in 22 reversed-phase chromatographic peaks, and for 17 products of 7,10-DMBAC found in 19 chromatographic peaks. The more abundant metabolites were derived from oxidation of the methyl groups. Other metabolites were dihydrodiols, epoxides, phenols and secondary metabolites. The 9-methyl group prevented dihydrodiol formation at the 8,9-position from 7,9-DMBAC, and for each carcinogen, the 3,4-dihydrodiol was formed. As well, 3,4-dihydrodiols of methyl oxidized compounds were found.

Morphine metabolites

DEFF Research Database (Denmark)

Christrup, Lona Louring

1997-01-01

, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) are the major metabolites of morphine. The metabolism of morphine occurs not only in the liver, but may also take place in the brain and the kidneys. The glucuronides are mainly eliminated via bile and urine. Glucuronides as a rule...... are considered as highly polar metabolites unable to cross the blood-brain barrier. Although morphine glucuronidation has been demonstrated in human brain tissue, the capacity is very low compared to that of the liver, indicating that the M3G and M6G concentrations observed in the cerebrospinal fluid (CSF) after...... systemic administration reflect hepatic metabolism of morphine and that the morphine glucuronides, despite their high polarity, can penetrate into the brain. Like morphine, M6G has been shown to be relatively more selective for mu-receptors than for delta- and kappa-receptors while M3G does not appear...

Prediction of Non-Equilibrium Kinetics of Fuel-Rich Kerosene/LOX Combustion in Gas Generator

International Nuclear Information System (INIS)

Yu, Jung Min; Lee, Chang Jin

2007-01-01

Gas generator is the device to produce high enthalpy gases needed to drive turbo-pump system in liquid rocket engine. And, the combustion temperature in gas generator should be controlled below around 1,000K to avoid any possible thermal damages to turbine blade by using either fuel rich combustion or oxidizer rich combustion. Thus, nonequilibrium chemical reaction dominates in fuel-rich combustion of gas generator. Meanwhile, kerosene is a compounded fuel with various types of hydrocarbon elements and difficult to model the chemical kinetics. This study focuses on the prediction of the non-equilibrium reaction of fuel rich kerosene/LOX combustion with detailed kinetics developed by Dagaut using PSR (Perfectly Stirred Reactor) assumption. In Dagaut's surrogate model for kerosene, chemical kinetics of kerosene consists of 1,592 reaction steps with 207 chemical species. Also, droplet evaporation time is taken into account in the PSR calculation by changing the residence time of droplet in the gas generator. Frenklach's soot model was implemented along with detailed kinetics to calculate the gas properties of fuel rich combustion efflux. The results could provide very reliable and accurate numbers in the prediction of combustion gas temperature,species fraction and material properties

Biotransformation of primary nicotine metabolites. I. In vivo metabolism of R-(+)-[14C-NCH3]N-methylnicotinium ion in the guinea pig

International Nuclear Information System (INIS)

Pool, W.F.; Crooks, P.A.

1985-01-01

The in vivo biotransformation and tissue distribution of the methylated nicotine metabolite R-(+)-[ 14 C-NCH 3 ]N-methylnicotinium acetate was studied in the guinea pig. The detection and quantification of 24-hr urinary metabolites after ip injection was determined by cation-exchange HPLC interfaced to a radiochemical flowthrough detector. The urinary metabolite profile consisted of five peaks. One eluted close to the void, and three coeluted with authentic standards of N-methylcotininium ion, N-methylnornicotinium ion, and N-methylnicotinium ion. A fifth, and as yet unidentified, metabolite was also detected. Tissue distribution of 14 C label after 24 hr was highest in the adrenal gland and epididymis followed by the gallbladder, bladder, kidney, spleen, and heart. No significant amounts of 14 C were found in the brain. The results indicate that N-methylcotininium ion and N-methylnornicotinium ion are both formed subsequent to the formation of N-methylnicotinium ion in the metabolism of R-(+)-nicotine in the guinea pig

NMR analysis of male fathead minnow urinary metabolites: A potential approach for studying impacts of chemical exposures

Energy Technology Data Exchange (ETDEWEB)

Ekman, D.R. [Ecosystems Research Division, U.S. EPA, 960 College Station Road, Athens, GA 30605 (United States)], E-mail: ekman.drew@epa.gov; Teng, Q. [Ecosystems Research Division, U.S. EPA, 960 College Station Road, Athens, GA 30605 (United States); Jensen, K.M.; Martinovic, D.; Villeneuve, D.L.; Ankley, G.T. [Mid-Continent Ecology Division, U.S. EPA, 6201 Congdon Boulevard, Duluth, MN 55804 (United States); Collette, T.W. [Ecosystems Research Division, U.S. EPA, 960 College Station Road, Athens, GA 30605 (United States)

2007-11-30

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D {sup 1}H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D {sup 1}H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of {sup 1}H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 {mu}g/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish.

NMR analysis of male fathead minnow urinary metabolites: A potential approach for studying impacts of chemical exposures

International Nuclear Information System (INIS)

Ekman, D.R.; Teng, Q.; Jensen, K.M.; Martinovic, D.; Villeneuve, D.L.; Ankley, G.T.; Collette, T.W.

2007-01-01

The potential for profiling metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy was used for the assignment of metabolites in urine from unexposed fish. Because fathead minnow urine is dilute, we lyophilized these samples prior to analysis. Furthermore, 1D 1 H NMR spectra of unlyophilized urine from unexposed male fathead minnow and Sprague-Dawley rat were acquired to qualitatively compare rat and fish metabolite profiles and to provide an estimate of the total urinary metabolite pool concentration difference. As a small proof-of-concept study, lyophilized urine samples from male fathead minnows exposed to three different concentrations of the antiandrogen vinclozolin were analyzed by 1D 1 H NMR to assess exposure-induced changes. Through a combination of principal components analysis (PCA) and measurements of 1 H NMR peak intensities, several metabolites were identified as changing with statistical significance in response to exposure. Among those changes occurring in response to exposure to the highest concentration (450 μg/L) of vinclozolin were large increases in taurine, lactate, acetate, and formate. These increases coincided with a marked decrease in hippurate, a combination potentially indicative of hepatotoxicity. The results of these investigations clearly demonstrate the potential utility of an NMR-based approach for assessing chemical exposures in male fathead minnow, using urine collected from individual fish

Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

KAUST Repository

Radogna, Flavia

2015-03-01

Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

(1) H-MRS processing parameters affect metabolite quantification

DEFF Research Database (Denmark)

Bhogal, Alex A; Schür, Remmelt R; Houtepen, Lotte C

2017-01-01

investigated the influence of model parameters and spectral quantification software on fitted metabolite concentration values. Sixty spectra in 30 individuals (repeated measures) were acquired using a 7-T MRI scanner. Data were processed by four independent research groups with the freedom to choose their own...... + NAAG/Cr + PCr and Glu/Cr + PCr, respectively. Metabolite quantification using identical (1) H-MRS data was influenced by processing parameters, basis sets and software choice. Locally preferred processing choices affected metabolite quantification, even when using identical software. Our results......Proton magnetic resonance spectroscopy ((1) H-MRS) can be used to quantify in vivo metabolite levels, such as lactate, γ-aminobutyric acid (GABA) and glutamate (Glu). However, there are considerable analysis choices which can alter the accuracy or precision of (1) H-MRS metabolite quantification...

Inhibition of Lysyl Oxidases Impairs Migration and Angiogenic Properties of Tumor-Associated Pericytes

Directory of Open Access Journals (Sweden)

Aline Lopes Ribeiro

2017-01-01

Full Text Available Pericytes are important cellular components of the tumor microenviroment with established roles in angiogenesis and metastasis. These two cancer hallmarks are modulated by enzymes of the LOX family, but thus far, information about LOX relevance in tumor-associated pericytes is lacking. Here, we performed a comparative characterization of normal and tumoral pericytes and report for the first time the modulatory effects of LOX enzymes on activated pericyte properties. Tumoral pericytes isolated from childhood ependymoma and neuroblastoma specimens displayed angiogenic properties in vitro and expressed typical markers, including CD146, NG2, and PDGFRβ. Expression of all LOX family members could be detected in both normal and tumor-associated pericytes. In most pericyte samples, LOXL3 was the family member displaying the highest transcript levels. Inhibition of LOX/LOXL activity with the inhibitor β-aminopropionitrile (βAPN significantly reduced migration of pericytes, while proliferation rates were kept unaltered. Formation of tube-like structures in vitro by pericytes was also significantly impaired upon inhibition of LOX/LOXL activity with βAPN, which induced more prominent effects in tumor-associated pericytes. These findings reveal a novel involvement of the LOX family of enzymes in migration and angiogenic properties of pericytes, with implications in tumor development and in therapeutic targeting tumor microenvironment constituents.

Pharmacokinetics of ketamine and its metabolite norketamine administered at a sub-anesthetic dose together with xylazine to calves prior to castration.

Science.gov (United States)

Gehring, R; Coetzee, J F; Tarus-Sang, J; Apley, M D

2009-04-01

The objective of this study was to evaluate the plasma pharmacokinetics of ketamine and its active metabolite norketamine administered intravenously at a dose of 0.1 mg/kg together with xylazine (0.05 mg/kg) to control the pain associated with castration in calves. A two-compartment model with an additional metabolite compartment linked to the central compartment was used to simultaneously describe the time-concentration profiles of both ketamine and its major metabolite norketamine. Parameter values estimated from the time-concentration profiles observed in this study were volume of the central compartment (V(c) = 132.82 +/- 68.23 mL/kg), distribution clearance (CL(D) = 15.49 +/- 2.56 mL/min/kg), volume of the peripheral compartment (V(T) = 257.05 +/- 41.65 mL/kg), ketamine clearance by the formation of the norketamine metabolite (CL(2M) = 8.56 +/- 7.37 mL/kg/min) and ketamine clearance by other routes (CL(o) = 16.41 +/- 3.42 mL/kg/min). Previously published data from rats suggest that the metabolite norketamine contributes to the analgesic effect of ketamine, with a potency that is one-third of the parent drug. An understanding of the time-concentration relationships and the disposition of the parent drug and its metabolite is therefore important for a better understanding of the analgesic potential of ketamine in cattle.

Cytotoxicity of pyrrolizidine alkaloid in human hepatic parenchymal and sinusoidal endothelial cells: Firm evidence for the reactive metabolites mediated pyrrolizidine alkaloid-induced hepatotoxicity.

Science.gov (United States)

Yang, Mengbi; Ruan, Jianqing; Fu, Peter P; Lin, Ge

2016-01-05

Pyrrolizidine alkaloids (PAs) widely distribute in plants and can cause hepatic sinusoidal obstruction syndrome (HSOS), which typically presents as a primary sinusoidal endothelial cell damage. It is well-recognized that after ingestion, PAs undergo hepatic cytochromes P450 (CYPs)-mediated metabolic activation to generate dehydropyrrolizidine alkaloids (DHPAs), which are hydrolyzed to dehydroretronecine (DHR). DHPAs and DHR are reactive metabolites having same core pyrrole moiety, and can bind proteins to form pyrrole-protein adducts, which are believed as the primary cause for PA-induced HSOS. However, to date, the direct evidences supporting the toxicity of DHPAs and DHR in the liver, in particular in the sinusoidal endothelial cells, are lacking. Using human hepatic sinusoidal endothelial cells (HSEC) and HepG2 (representing hepatic parenchymal cells), cells that lack CYPs activity, this study determined the direct cytotoxicity of dehydromonocrotaline, a representative DHPA, and DHR, but no cytotoxicity of the intact PA (monocrotaline) in both cell lines, confirming that reactive metabolites mediate PA intoxication. Comparing with HepG2, HSEC had significantly lower basal glutathione (GSH) level, and was significantly more susceptible to the reactive metabolites with severer GSH depletion and pyrrole-protein adducts formation. The toxic potency of two reactive metabolites was also compared. DHPA was more reactive than DHR, leading to severer toxicity. In conclusion, our results unambiguously provided the first direct evidence for the critical role of DHPA and DHR in the reactive metabolites-mediated PA-induced hepatotoxicity, which occurs predominantly in HSEC due to severe GSH depletion and the significant formation of pyrrole-protein adducts in HSEC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Science.gov (United States)

Schadt, Simone; Bister, Bojan; Chowdhury, Swapan K; Funk, Christoph; Hop, Cornelis E C A; Humphreys, W Griffith; Igarashi, Fumihiko; James, Alexander D; Kagan, Mark; Khojasteh, S Cyrus; Nedderman, Angus N R; Prakash, Chandra; Runge, Frank; Scheible, Holger; Spracklin, Douglas K; Swart, Piet; Tse, Susanna; Yuan, Josh; Obach, R Scott

2018-06-01

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

GPCR-Mediated Signaling of Metabolites

DEFF Research Database (Denmark)

Husted, Anna Sofie; Trauelsen, Mette; Rudenko, Olga

2017-01-01

microbiota target primarily enteroendocrine, neuronal, and immune cells in the lamina propria of the gut mucosa and the liver and, through these tissues, the rest of the body. In contrast, metabolites from the intermediary metabolism act mainly as metabolic stress-induced autocrine and paracrine signals...... and obesity. The concept of key metabolites as ligands for specific GPCRs has broadened our understanding of metabolic signaling significantly and provides a number of novel potential drug targets....

7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

Science.gov (United States)

He, Xiaobo; Xia, Qingsu; Ma, Liang; Fu, Peter P

2016-01-01

Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

The relevance of "non-relevant metabolites" from plant protection products (PPPs) for drinking water: the German view.

Science.gov (United States)

Dieter, Hermann H

2010-03-01

"Non-relevant metabolites" are those degradation products of plant protection products (PPPs), which are devoid of the targeted toxicities of the PPP and devoid of genotoxicity. Most often, "non-relevant metabolites" have a high affinity to the aquatic environment, are very mobile within this environment, and, usually, are also persistent. Therefore, from the point of drinking water hygiene, they must be characterized as "relevant for drinking water" like many other hydrophilic/polar environmental contaminants of different origins. "Non-relevant metabolites" may therefore penetrate to water sources used for abstraction of drinking water and may thus ultimately be present in drinking water. The presence of "non-relevant metabolites" and similar trace compounds in the water cycle may endanger drinking water quality on a long-term scale. During oxidative drinking water treatment, "non-relevant metabolites" may also serve as the starting material for toxicologically relevant transformation products similar to processes observed by drinking water disinfection with chlorine. This hypothesis was recently confirmed by the detection of the formation of N-nitroso-dimethylamine from ozone and dimethylsulfamide, a "non-relevant metabolite" of the fungicide tolylfluanide. In order to keep drinking water preferably free of "non-relevant metabolites", the German drinking water advisory board of the Federal Ministry of Health supports limiting their penetration into raw and drinking water to the functionally (agriculturally) unavoidable extent. On this background, the German Federal Environment Agency (UBA) recently has recommended two health related indication values (HRIV) to assess "non-relevant metabolites" from the view of drinking water hygiene. Considering the sometimes incomplete toxicological data base for some "non-relevant metabolites", HRIV also have the role of health related precautionary values. Depending on the completeness and quality of the toxicological

Facilitated uptake of a bioactive metabolite of maritime pine bark extract (pycnogenol into human erythrocytes.

Directory of Open Access Journals (Sweden)

Max Kurlbaum

Full Text Available Many plant secondary metabolites exhibit some degree of biological activity in humans. It is a common observation that individual plant-derived compounds in vivo are present in the nanomolar concentration range at which they usually fail to display measurable activity in vitro. While it is debatable that compounds detected in plasma are not the key effectors of bioactivity, an alternative hypothesis may take into consideration that measurable concentrations also reside in compartments other than plasma. We analysed the binding of constituents and the metabolite δ-(3,4-dihydroxy-phenyl-γ-valerolactone (M1, that had been previously detected in plasma samples of human consumers of pine bark extract Pycnogenol, to human erythrocytes. We found that caffeic acid, taxifolin, and ferulic acid passively bind to red blood cells, but only the bioactive metabolite M1 revealed pronounced accumulation. The partitioning of M1 into erythrocytes was significantly diminished at higher concentrations of M1 and in the presence of glucose, suggesting a facilitated transport of M1 via GLUT-1 transporter. This concept was further supported by structural similarities between the natural substrate α-D-glucose and the S-isomer of M1. After cellular uptake, M1 underwent further metabolism by conjugation with glutathione. We present strong indication for a transporter-mediated accumulation of a flavonoid metabolite in human erythrocytes and subsequent formation of a novel glutathione adduct. The physiologic role of the adduct remains to be elucidated.

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes

Directory of Open Access Journals (Sweden)

Schrey Silvia D

2012-08-01

Full Text Available Abstract Background Studies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner. Results Fifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum. The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol and siderophores (e.g. ferulic acid, desferroxiamines. Plant disease resistance was activated by a single streptomycete strain only. Conclusions Mycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites.

Glucose metabolite glyoxal induces senescence in telomerase-immortalized human mesenchymal stem cells

DEFF Research Database (Denmark)

Larsen, Simon Asbjørn; Kassem, Moustapha; Rattan, Suresh

2012-01-01

). Furthermore, the in vitro differentiation potential of hMSC-TERT to become functional osteoblasts was highly reduced in GO-treated stem cells, as determined by alkaline phosphatase (ALP) activity and mineralized matrix (MM) formation. Conclusions The results of our study imply that an imbalanced glucose...... physiological metabolite produced by the auto-oxidation of glucose, and can form covalent adducts known as advanced glycation endproducts (AGE). We have previously reported that GO accelerates ageing and causes premature senescence in normal human skin fibroblasts. Results Using a bone marrow-derived telomerase...

21 CFR 862.3250 - Cocaine and cocaine metabolite test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cocaine and cocaine metabolite test system. 862... Test Systems § 862.3250 Cocaine and cocaine metabolite test system. (a) Identification. A cocaine and cocaine metabolite test system is a device intended to measure cocaine and a cocaine metabolite...

Urinary excretion of androgen metabolites, comparison with excretion of radioactive metabolites after injection of (4-/sup 14/C)testosterone. Influence of age

Energy Technology Data Exchange (ETDEWEB)

Deslypere, J P; Sayed, A; Vermeulen, A [Department of Internal Medicine, Section of Endocrinology, State University Academic Hospital, De Pintelaan, 135, Ghent, Belgium; Wiers, P W [Department of Internal Medicine, Section of Pneumology, State University Academic Hospital, The Netherlands

1981-01-01

The influence of age on the metabolic pattern of (4-/sup 14/C)testosterone was studied in 20 young and 8 elderly males and compared to the metabolic pattern of endogenous androgens; the latter was also studied in 16 young and 8 elderly women. In both young and elderly males, androsterone and aetiocholanolone glucuronide represent 65% of (4-/sup 14/C)testosterone metabolites: together with their suephoconjugates as well as with 5..cap alpha..- and 5..beta..-androstane-3..cap alpha.., 17..beta..-diol they represent even more than 75% of total urinary metabolites. The 5..cap alpha../5..beta.. ratio of metabolites of (4-/sup 14/C)testosterone was significantly (P<0.01) correlated with the 5..cap alpha../5..beta.. ratio of the metabolites of the endogenous androgens, mainly dehydroepiandrosterone and androstenedione. The 5..cap alpha../5..beta.. ratio of (4-/sup 14/C)testosterone metabolites was generally higher than the ratio of metabolites of endogenous androgens, suggesting that the transformation of T to ring A saturated metabolites occurs at least partially in another compartment than the transformation of DHEA to these metabolites. For both (4-/sup 14/C)testosterone and endogenous androgen metabolites we observed a statistically significant reduction of the 5..cap alpha../5..beta.. ratio with age, a general phenomenon in both males and females. This reduction concern also 11-OH-androst-4-ene-3.17-dione metabolism. Neither sex hormone levels, nor specific binding seems to determine this age dependent shift; neither is there convincing evidence for latent hypothyroisism or liver dysfunction in the elderly. An age associated primary decrease of the 5..cap alpha..-reductase activity seems the most likely explanation.

SECONDARY METABOLITES FROM MARINE PENICILLIUM BREVICOMPACTUM

OpenAIRE

ROVIROSA, JUANA; DIAZ-MARRERO, ANA; DARIAS, JOSE; PAINEMAL, KARIN; SAN MARTIN, AURELIO

2006-01-01

In a screening of Basidiomycete cultures isolated from marine invertebrates collected along the Chilean coastline for the production of antibiotics we identified a Penicillium brevicompactum strain as a producer of metabolites inhibiting the growth of bacteria and fungi. Bioactivity guided purification resulted in the isolation of four known metabolites. Their structures were elucidated by spectroscopic methods.

Plant metabolites and nutritional quality of vegetables.

Science.gov (United States)

Hounsome, N; Hounsome, B; Tomos, D; Edwards-Jones, G

2008-05-01

Vegetables are an important part of the human diet and a major source of biologically active substances such as vitamins, dietary fiber, antioxidants, and cholesterol-lowering compounds. Despite a large amount of information on this topic, the nutritional quality of vegetables has not been defined. Historically, the value of many plant nutrients and health-promoting compounds was discovered by trial and error. By the turn of the century, the application of chromatography, mass spectrometry, infrared spectrometry, and nuclear magnetic resonance allowed quantitative and qualitative measurements of a large number of plant metabolites. Approximately 50000 metabolites have been elucidated in plants, and it is predicted that the final number will exceed 200000. Most of them have unknown function. Metabolites such as carbohydrates, organic and amino acids, vitamins, hormones, flavonoids, phenolics, and glucosinolates are essential for plant growth, development, stress adaptation, and defense. Besides the importance for the plant itself, such metabolites determine the nutritional quality of food, color, taste, smell, antioxidative, anticarcinogenic, antihypertension, anti-inflammatory, antimicrobial, immunostimulating, and cholesterol-lowering properties. This review is focused on major plant metabolites that characterize the nutritional quality of vegetables, and methods of their analysis.

β-Orcinol Metabolites from the Lichen Hypotrachyna revoluta

Directory of Open Access Journals (Sweden)

Panagiota Papadopoulou

2007-05-01

Full Text Available Four new β-orcinol metabolites, hypotrachynic acid (1, deoxystictic acid (2, cryptostictinolide (3 and 8 ́-methylconstictic acid (4 along with the metabolites 8 ́-methylstictic acid (5, 8 ́-methylmenegazziaic acid (6, stictic acid (7, 8 ́-ethylstictic acid (8 and atranorin (9, that have been previously described, were isolated for the first time from the tissue extracts of the lichen Hypotrachyna revoluta (Flörke Hale. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analyses. Radical scavenging activity (RSA of the metabolites isolated in adequate amounts, was evaluated using luminol chemiluminescence and comparison with Trolox®.

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols.

Science.gov (United States)

Pinu, Farhana R; Villas-Boas, Silas G; Aggio, Raphael

2017-10-23

Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

Detecting beer intake by unique metabolite patterns

DEFF Research Database (Denmark)

Gürdeniz, Gözde; Jensen, Morten Georg; Meier, Sebastian

2016-01-01

Evaluation of health related effects of beer intake is hampered by the lack of accurate tools for assessing intakes (biomarkers). Therefore, we identified plasma and urine metabolites associated with recent beer intake by untargeted metabolomics and established a characteristic metabolite pattern...... representing raw materials and beer production as a qualitative biomarker of beer intake. In a randomized, crossover, single-blinded meal study (MSt1) 18 participants were given one at a time four different test beverages: strong, regular and non-alcoholic beers and a soft drink. Four participants were...... assigned to have two additional beers (MSt2). In addition to plasma and urine samples, test beverages, wort and hops extract were analyzed by UPLC-QTOF. A unique metabolite pattern reflecting beer metabolome, including metabolites derived from beer raw material (i.e. N-methyl tyramine sulfate and the sum...

Metabolic changes associated with shoot formation in tobacco callus cultures

Energy Technology Data Exchange (ETDEWEB)

Grady, K.L.

1982-08-01

Callus tissue derived from Nicotiana tabacum L. stem pith parenchyma cells was grown either on medium which maintains the callus in an undifferentiated state, or on medium which induces the formation of shoots. Two complementary types of studies were performed with the goal of establishing metabolic markers for the initiation of shoot formation: one designed to characterize the flow of radioactive sucrose into various metabolic pools, and one which allowed measurement of intermediary metabolite concentrations. In the former, callus tissue was incubated in (U-/sup 14/C)sucrose for periods up to one hour, and patterns of metabolite labelling in tissue grown on shoot-forming and non-shoot-forming media were compared. In the latter studies, tissue was grown for an entire subculture period on non-shoot-forming medium labelled with (U-/sup 14/C)sucrose, then subcultured to labelled non-shoot-forming or shoot-forming media, and sampled at intervals during the first week of growth. 189 references.

Metabolic changes associated with shoot formation in tobacco callus cultures

International Nuclear Information System (INIS)

Grady, K.L.

1982-08-01

Callus tissue derived from Nicotiana tabacum L. stem pith parenchyma cells was grown either on medium which maintains the callus in an undifferentiated state, or on medium which induces the formation of shoots. Two complementary types of studies were performed with the goal of establishing metabolic markers for the initiation of shoot formation: one designed to characterize the flow of radioactive sucrose into various metabolic pools, and one which allowed measurement of intermediary metabolite concentrations. In the former, callus tissue was incubated in [U- 14 C]sucrose for periods up to one hour, and patterns of metabolite labelling in tissue grown on shoot-forming and non-shoot-forming media were compared. In the latter studies, tissue was grown for an entire subculture period on non-shoot-forming medium labelled with [U- 14 C]sucrose, then subcultured to labelled non-shoot-forming or shoot-forming media, and sampled at intervals during the first week of growth. 189 references

Plutonium interactions with soil microbial metabolites: effect on plutonium sorption by soil

International Nuclear Information System (INIS)

Wildung, R.E.; Garland, T.R.; Rogers, J.E.

1987-01-01

To develop an understanding of the mechanisms of plutonium (Pu) complexation and solubilization by soil microorganisms, a broad range of bacteria and fungi were isolated in pure cultures from soil on the basis of metal tolerance and carbon requirements. The organisms were then used in investigations to examine Pu cellular transport, Pu complexation by extracellular metabolites, and the effects of complexation on Pu valence state, chemical form, and solubility in soil. Of the 239 bacteria and 250 fungi isolated from soil, 19 bacteria and 60 fungi were selected for detailed study. Of these organisms, 15 bacteria and 18 fungi grew to form extracellular Pu complexes that increased the concentration of Pu in soil column eluates relative to controls. Elution through soil effectively removed positively charged Pu complexes. Increased Pu mobility in soil resulted from the formation of neutral and negatively charged Pu complexes, which differed with organism type. In the presence of known microbial metabolites and synthetic ligands (DTPA, EDTA, EDDHA), Pu(VI) was reduced to Pu(IV) before complexation, suggesting that Pu(IV) would be the dominant valence state associated with organic complexes in soils. Studies on selected organisms indicated that both active Pu transport and Pu sorption on the cell occurred, and these phenomena, as well as complexation by extracellular metabolites of Pu, were a function of the form of Pu supplied, the organism type and growth characteristics, and the ability of the organism to alter extracellular pH. 18 references, 6 figures, 7 tables

Marine metabolites: The sterols of soft coral

Digital Repository Service at National Institute of Oceanography (India)

Sarma, N.S.; Krishna, M.S.; Pasha, Sk.G.; Rao, T.S.P.; Venkateswarlu, Y.; Parameswaran, P.S.

Sterols constitute a major group of secondary metabolites of soft corals. Several of these compounds have the 'usual' 3 beta-hydroxy, delta sup(5) (or delta sup(0)) cholestane skeleton, a large number of these metabolites are polar sterols...

MIDAS: a database-searching algorithm for metabolite identification in metabolomics.

Science.gov (United States)

Wang, Yingfeng; Kora, Guruprasad; Bowen, Benjamin P; Pan, Chongle

2014-10-07

A database searching approach can be used for metabolite identification in metabolomics by matching measured tandem mass spectra (MS/MS) against the predicted fragments of metabolites in a database. Here, we present the open-source MIDAS algorithm (Metabolite Identification via Database Searching). To evaluate a metabolite-spectrum match (MSM), MIDAS first enumerates possible fragments from a metabolite by systematic bond dissociation, then calculates the plausibility of the fragments based on their fragmentation pathways, and finally scores the MSM to assess how well the experimental MS/MS spectrum from collision-induced dissociation (CID) is explained by the metabolite's predicted CID MS/MS spectrum. MIDAS was designed to search high-resolution tandem mass spectra acquired on time-of-flight or Orbitrap mass spectrometer against a metabolite database in an automated and high-throughput manner. The accuracy of metabolite identification by MIDAS was benchmarked using four sets of standard tandem mass spectra from MassBank. On average, for 77% of original spectra and 84% of composite spectra, MIDAS correctly ranked the true compounds as the first MSMs out of all MetaCyc metabolites as decoys. MIDAS correctly identified 46% more original spectra and 59% more composite spectra at the first MSMs than an existing database-searching algorithm, MetFrag. MIDAS was showcased by searching a published real-world measurement of a metabolome from Synechococcus sp. PCC 7002 against the MetaCyc metabolite database. MIDAS identified many metabolites missed in the previous study. MIDAS identifications should be considered only as candidate metabolites, which need to be confirmed using standard compounds. To facilitate manual validation, MIDAS provides annotated spectra for MSMs and labels observed mass spectral peaks with predicted fragments. The database searching and manual validation can be performed online at http://midas.omicsbio.org.

Dynamic microbial succession of Shanxi aged vinegar and its correlation with flavor metabolites during different stages of acetic acid fermentation.

Science.gov (United States)

Zhu, Yunping; Zhang, Feifei; Zhang, Chengnan; Yang, Li; Fan, Guangsen; Xu, Youqiang; Sun, Baoguo; Li, Xiuting

2018-06-05

Shanxi aged vinegar (SAV), one of the famous Chinese vinegars, is produced by multispecies solid-state fermentation in which the acetic acid fermentation stage (AAF) is especially important. However, how bacterial succession and their metabolites change along with the different stages of AAF is still poorly understood. In this study, we investigated the dynamic bacterial succession and flavor formation in three batches of SAV using high-throughput sequencing and metabolomics approaches. It is interesting to find that AAF can be divided into three stages based on its bacterial community succession (early stage, days 0-4; medium stage, days 5-21; and later stage, days 22-26). Pantoea, Pediococcus, Lactococcus and Rhizobium played an important role in the early stage; Lactobacillus was dominant in the medium stage (67.72%); and Acetobacter, Komagataeibacter and Kroppenstedtia were the key bacteria in the later stage. A total of seven organic acids and 42 volatile constituents (esters, alcohol, ketones and aldehydes) were detected during the AAF. Spearman correlation analysis showed a significant correlation between the bacterial community and these flavor metabolites during the AAF of the SAV. This is the first report to explore the relationships between volatile flavor metabolites and bacterial community succession by a three-staged method and provide theoretical support for a flavor formation mechanism in traditional SAV.

Effects of aspartame metabolites on astrocytes and neurons.

Science.gov (United States)

Rycerz, Karol; Jaworska-Adamu, Jadwiga Elżbieta

2013-01-01

Aspartame, a widespread sweetener used in many food products, is considered as a highly hazardous compound. Aspartame was discovered in 1965 and raises a lot of controversy up to date. Astrocytes are glial cells, the presence and functions of which are closely connected with the central nervous system (CNS). The aim of this article is to demonstrate the direct and indirect role of astrocytes participating in the harmful effects of aspartame metabolites on neurons. The artificial sweetener is broken down into phenylalanine (50%), aspartic acid (40%) and methanol (10%) during metabolism in the body. The excess of phenylalanine blocks the transport of important amino acids to the brain contributing to reduced levels of dopamine and serotonin. Astrocytes directly affect the transport of this amino acid and also indirectly by modulation of carriers in the endothelium. Aspartic acid at high concentrations is a toxin that causes hyperexcitability of neurons and is also a precursor of other excitatory amino acid - glutamates. Their excess in quantity and lack of astrocytic uptake induces excitotoxicity and leads to the degeneration of astrocytes and neurons. The methanol metabolites cause CNS depression, vision disorders and other symptoms leading ultimately to metabolic acidosis and coma. Astrocytes do not play a significant role in methanol poisoning due to a permanent consumption of large amounts of aspartame. Despite intense speculations about the carcinogenicity of aspartame, the latest studies show that its metabolite - diketopiperazine - is cancirogenic in the CNS. It contributes to the formation of tumors in the CNS such as gliomas, medulloblastomas and meningiomas. Glial cells are the main source of tumors, which can be caused inter alia by the sweetener in the brain. On the one hand the action of astrocytes during aspartame poisoning may be advantageous for neuro-protection while on the other it may intensify the destruction of neurons. The role of the glia in

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

Science.gov (United States)

Bist, Itti; Bhakta, Snehasis; Jiang, Di; Keyes, Tia E; Martin, Aaron; Forster, Robert J; Rusling, James F

2017-11-21

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S N 2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu 2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 6 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD 50 and Comet assay results.

40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and... degradation of less than 10 percent in a 30-day period. (b) Contaminants and impurities. The presence in any...

Analysis of Intracellular Metabolites from Microorganisms: Quenching and Extraction Protocols

Directory of Open Access Journals (Sweden)

Farhana R. Pinu

2017-10-01

Full Text Available Sample preparation is one of the most important steps in metabolome analysis. The challenges of determining microbial metabolome have been well discussed within the research community and many improvements have already been achieved in last decade. The analysis of intracellular metabolites is particularly challenging. Environmental perturbations may considerably affect microbial metabolism, which results in intracellular metabolites being rapidly degraded or metabolized by enzymatic reactions. Therefore, quenching or the complete stop of cell metabolism is a pre-requisite for accurate intracellular metabolite analysis. After quenching, metabolites need to be extracted from the intracellular compartment. The choice of the most suitable metabolite extraction method/s is another crucial step. The literature indicates that specific classes of metabolites are better extracted by different extraction protocols. In this review, we discuss the technical aspects and advancements of quenching and extraction of intracellular metabolite analysis from microbial cells.

Investigation of metabolites for estimating blood deposition time.

Science.gov (United States)

Lech, Karolina; Liu, Fan; Davies, Sarah K; Ackermann, Katrin; Ang, Joo Ern; Middleton, Benita; Revell, Victoria L; Raynaud, Florence J; Hoveijn, Igor; Hut, Roelof A; Skene, Debra J; Kayser, Manfred

2018-01-01

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor's alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

The Role of Nitric Oxide Metabolites in the Formation of Endothelial Dysfunction in Children with Persistent Bronchial Asthma

Directory of Open Access Journals (Sweden)

Yu.V. Odinets

2016-09-01

Full Text Available The aim of the study was to assess the role of nitric oxide metabolites in the development of endothelium dysfunction in children with persistent bronchial asthma in acute period and during remission Material and methods. Fifty eight children aged 6–17 years old suffering from persistent bronchial asthma in the period of exacerbation and remission of disease and 15 healthy children of control group have been exa­mined in order to evaluate the role of the metabolites of nitric oxide levels for the development of endothelium dysfunction in the children with persistent bronchial asthma. There were determined serum blood S-nitrosothiol, NO2, NO3, the intima-media complex by the ultrasound duplex scanning and endothelium-dependent dilation status of the brachial artery by cuff probe. Results. It showed a reduction in the levels of nitric oxide metabolites and endothelium-dependent dilation of the brachial artery, intima-media thickening in the period of exacerbation. There was revealed a significant increase in the levels of nitric oxide metabolites and endothelium-dependent dilation of the brachial artery compared with the period of exacerbation, but these findings are still lower in comparison with the control group, which indicates the presence of persistent changes in the vascular wall. Conclusion. The nitric oxide metabolites as well as the carotid intimal medial thickness and endothelium-dependent dilation of brachial artery should be marders of endothelium dysfunction in the children with bronchial asthma.

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

Science.gov (United States)

Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

2011-02-03

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

Ruta graveolens Extracts and Metabolites against Spodoptera frugiperda.

Science.gov (United States)

Ayil-Gutiérrez, Benjamin A; Villegas-Mendoza, Jesús M; Santes-Hernndez, Zuridai; Paz-González, Alma D; Mireles-Martínez, Maribel; Rosas-García, Ninfa M; Rivera, Gildardo

2015-11-01

The biological activity of Ruta graveolens leaf tissue extracts obtained with different solvents (ethyl acetate, ethanol, and water) and metabolites (psoralen, 2- undecanone and rutin) against Spodoptera frugiperda was evaluated. Metabolites levels in extracts were quantified by HPLC and GC. Ethyl acetate and ethanol extracts showed 94% and 78% mortality, respectively. Additionally, psoralen metabolite showed a high mortality as cypermethrin. Metabolite quantification in extracts shows the presence of 2-undecanone (87.9 µmoles mg(-1) DW), psoralen (3.6 µmoles mg(-1) DW) and rutin (0.001 pmoles mg(-1) DW). We suggest that these concentrations of 2-undecanone and psoralen in R. graveolens leaf tissue extracts could be responsible for S. frugiperda mortality.

A nitrous acid biosynthetic pathway for diazo group formation in bacteria.

Science.gov (United States)

Sugai, Yoshinori; Katsuyama, Yohei; Ohnishi, Yasuo

2016-02-01

Although some diazo compounds have bioactivities of medicinal interest, little is known about diazo group formation in nature. Here we describe an unprecedented nitrous acid biosynthetic pathway responsible for the formation of a diazo group in the biosynthesis of the ortho-diazoquinone secondary metabolite cremeomycin in Streptomyces cremeus. This finding provides important insights into the biosynthetic pathways not only for diazo compounds but also for other naturally occurring compounds containing nitrogen-nitrogen bonds.

Direct coupling of electromembrane extraction to mass spectrometry - Advancing the probe functionality toward measurements of zwitterionic drug metabolites.

Science.gov (United States)

Rye, Torstein Kige; Fuchs, David; Pedersen-Bjergaard, Stig; Petersen, Nickolaj Jacob

2017-08-29

A triple-flow electromembrane extraction (EME) probe was developed and coupled directly to electrospray-ionization mass spectrometry (ESI-MS). Metabolic reaction mixtures (pH 7.4) containing drug substances and related metabolites were continuously drawn (20 μL/min) into the EME probe in one flow channel, and mixed inside the probe with 7.5 μL min -1 of 1 M formic acid as make-up flow from a second flow channel. Following this acidification, the drug substances and their related metabolites were continuously extracted by EME at 400 V, across a supported liquid membrane (SLM) comprising 2-nitrophenyl octyl ether (and for some experiments containing 30% triphenyl phosphate (TPP)), and into 20 μL min -1 of formic acid as acceptor phase, which was introduced through a third flow channel. The acceptor phase was pumped directly to the MS system, and the ion intensity of extracted analytes was followed continuously as function of time. The triple-flow EME probe was used for co-extraction of positively charged parent drugs and their zwitterionic drug metabolites (hydroxyzine and its carboxylic acid metabolite cetirizine; and vortioxetine and its carboxylic acid metabolite Lu AA34443). While the zwitterionic metabolites could not be extracted at pH 7.4, it was shown that by acidifying the sample solution the zwitterionic metabolites could be extracted effectively. Various extraction parameters like make-up flow, extraction voltage and SLM composition were optimized for simultaneous extraction of parent drugs and metabolites. It was found that TPP added to the SLM improved extraction efficiencies of certain drug metabolites. Finally the optimized and characterized triple-flow EME probe was used for online studying the in-vitro metabolism of hydroxyzine and vortioxetine by rat liver microsomes. Due to the automated pre-extraction acidification of the rat liver microsomal solutions, it was possible to continuously monitor formation of the zwitterionic drug

In vitro induction of tuber formation for the synthesis of secondary ...

African Journals Online (AJOL)

In vitro induction of tuber formation for the synthesis of secondary metabolites in Chlorophytum borivilianum Sant. et Fernand. Gulab S Thakur, Rohit Sharma, Bhagwan S Sanodiya, Rakesh Baghel, Radhika Thakur, Bansh N Singh, Ashish Savita, Avinash Dubey, Latasha Sikarwar, Pallavi Jaiswal, Gunja Khatri, GBKS ...

Participation of Arachidonic Acid Metabolism in the Aortic Aneurysm Formation in Patients with Marfan Syndrome

Directory of Open Access Journals (Sweden)

María E. Soto

2018-02-01

Full Text Available Marfan syndrome (MFS is a pleiotropic genetic disease involving the cardiovascular system where a fibrillin-1 mutation is present. This mutation is associated with accelerated activation of transforming growth factor β (TGFβ1 which contributes to the formation of aneurysms in the root of the aorta. There is an imbalance in the synthesis of thromboxane A2 (TXA2 and prostacyclin, that is a consequence of a differential protein expression of the isoforms of cyclooxygenases (COXs, suggesting an alteration of arachidonic acid (AA metabolism. The aim of this study was to analyze the participation of AA metabolism associated with inflammatory factors in the dilation and dissection of the aortic aneurysm in patients with MFS. A decrease in AA (p = 0.02, an increase in oleic acid (OA, TGFβ1, tumor necrosis factor alpha (TNFα, prostaglandin E2 (PGE2 (p < 0.05, and COXs activity (p = 0.002 was found. The expressions of phospholipase A2 (PLA2, cytochrome P450 (CYP450 4A, 5-lipoxygenase (5-LOX, COX2 and TXA2R (p < 0.05 showed a significant increase in the aortic aneurysm of patients with MFS compared to control subjects. COX1, 6-keto-prostaglandin 1 alpha (6-keto-PG1α and 8-isoprostane did not show significant changes. Histological examination of the aortas showed an increase of cystic necrosis, elastic fibers and collagen in MFS. The results suggest that there are inflammatory factors coupled to genetic factors that predispose to aortic endothelial dysfunction in the aortic tissue of patients with MFS. There is a decrease in the percentage of AA, associated with an increase of PLA2, COX2/TXA2R, CYP450 4A, and 5-LOX which leads to a greater synthesis of PGE2 than of 6-keto-PGF1α, thus contributing to the formation of the aortic aneurysm. The evident loss of the homeostasis in these mechanisms confirms that there is a participation of the AA pathway in the aneurysm progression in MFS.

Biochemical and secondary metabolites changes under moisture ...

African Journals Online (AJOL)

The study showed the importance of carbohydrate and nitrogen cycle related metabolites in mediating tolerance in cassava by affecting their phenotypic expression in the plant. Keywords: Hydrothermal stress, bio-chemicals, pigments, secondary metabolites, cassava. African Journal of Biotechnology, Vol 13(31) 3173-3186 ...

Hemizygous Le-Cre Transgenic Mice Have Severe Eye Abnormalities on Some Genetic Backgrounds in the Absence of LoxP Sites

Science.gov (United States)

Dorà, Natalie J.; Collinson, J. Martin; Hill, Robert E.; West, John D.

2014-01-01

Eye phenotypes were investigated in Le-CreTg/−; Pax6fl/+ mice, which were expected to show tissue-specific reduction of Pax6 in surface ectoderm derivatives. To provide a better comparison with our previous studies of Pax6+/− eye phenotypes, hemizygous Le-CreTg/− and heterozygous Pax6fl/+mice were crossed onto the CBA/Ca genetic background. After the Le-Cre transgene had been backcrossed to CBA/Ca for seven generations, significant eye abnormalities occurred in some hemizygous Le-CreTg/−; Pax6+/+ controls (without a floxed Pax6fl allele) as well as experimental Le-CreTg/−; Pax6fl/+ mice. However, no abnormalities were seen in Le-Cre−/−; Pax6fl/+ or Le-Cre−/−; Pax6+/+ controls (without the Le-Cre transgene). The severity and frequency of the eye abnormalities in Le-CreTg/−; Pax6+/+ control mice diminished after backcrossing Le-CreTg/− mice to the original FVB/N strain for two generations, showing that the effect was reversible. This genetic background effect suggests that the eye abnormalities are a consequence of an interaction between the Le-Cre transgene and alleles of unknown modifier genes present in certain genetic backgrounds. The abnormalities were also ameliorated by introducing additional Pax6 gene copies on a CBA/Ca background, suggesting involvement of Pax6 depletion in Le-CreTg/−; Pax6+/+ mice rather than direct action of Cre recombinase on cryptic pseudo-loxP sites. One possibility is that expression of Cre recombinase from the Pax6-Le regulatory sequences in the Le-Cre transgene depletes cofactors required for endogenous Pax6 gene expression. Our observation that eye abnormalities can occur in hemizygous Le-CreTg/−; Pax6+/+ mice, in the absence of a floxed allele, demonstrates the importance of including all the relevant genetic controls in Cre-loxP experiments. PMID:25272013

Prospective study of blood metabolites associated with colorectal cancer risk.

Science.gov (United States)

Shu, Xiang; Xiang, Yong-Bing; Rothman, Nathaniel; Yu, Danxia; Li, Hong-Lan; Yang, Gong; Cai, Hui; Ma, Xiao; Lan, Qing; Gao, Yu-Tang; Jia, Wei; Shu, Xiao-Ou; Zheng, Wei

2018-02-26

Few prospective studies, and none in Asians, have systematically evaluated the relationship between blood metabolites and colorectal cancer risk. We conducted a nested case-control study to search for risk-associated metabolite biomarkers for colorectal cancer in an Asian population using blood samples collected prior to cancer diagnosis. Conditional logistic regression was performed to assess associations of metabolites with cancer risk. In this study, we included 250 incident cases with colorectal cancer and individually matched controls nested within two prospective Shanghai cohorts. We found 35 metabolites associated with risk of colorectal cancer after adjusting for multiple comparisons. Among them, 12 metabolites were glycerophospholipids including nine associated with reduced risk of colorectal cancer and three with increased risk [odds ratios per standard deviation increase of transformed metabolites: 0.31-1.98; p values: 0.002-1.25 × 10 -10 ]. The other 23 metabolites associated with colorectal cancer risk included nine lipids other than glycerophospholipid, seven aromatic compounds, five organic acids and four other organic compounds. After mutual adjustment, nine metabolites remained statistically significant for colorectal cancer. Together, these independently associated metabolites can separate cancer cases from controls with an area under the curve of 0.76 for colorectal cancer. We have identified that dysregulation of glycerophospholipids may contribute to risk of colorectal cancer. © 2018 UICC.

MicroRNAs and Metabolites in Serum Change after Chemotherapy: Impact on Hematopoietic Stem and Progenitor Cells.

Directory of Open Access Journals (Sweden)

Thomas Walenda

Full Text Available Hematopoietic regeneration after high dose chemotherapy necessitates activation of the stem cell pool. There is evidence that serum taken after chemotherapy comprises factors stimulating proliferation and self-renewal of CD34(+ hematopoietic stem and progenitor cells (HSPCs--however, the nature of these feedback signals is yet unclear. Here, we addressed the question if specific microRNAs (miRNAs or metabolites are affected after high dose chemotherapy. Serum taken from the same patients before and after chemotherapy was supplemented for in vitro cultivation of HSPCs. Serum taken after chemotherapy significantly enhanced HSPC proliferation, better maintained a CD34(+ immunophenotype, and stimulated colony forming units. Microarray analysis revealed that 23 miRNAs changed in serum after chemotherapy--particularly, miRNA-320c and miRNA-1275 were down-regulated whereas miRNA-3663-3p was up-regulated. miRNA-320c was exemplarily inhibited by an antagomiR, which seemed to increase proliferation. Metabolomic profiling demonstrated that 44 metabolites were less abundant, whereas three (including 2-hydroxybutyrate and taurocholenate sulphate increased in serum upon chemotherapy. Nine of these metabolites were subsequently tested for effects on HSPCs in vitro, but none of them exerted a clear concentration dependent effect on proliferation, immunophenotype and colony forming unit formation. Taken together, serum profiles of miRNAs and metabolites changed after chemotherapy. Rather than individually, these factors may act in concert to recruit HSPCs into action for hematopoietic regeneration.

7-Glutathione-pyrrole and 7-cysteine-pyrrole are potential carcinogenic metabolites of pyrrolizidine alkaloids.

Science.gov (United States)

He, Xiaobo; Xia, Qingsu; Fu, Peter P

2017-04-03

Many pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Metabolism of PAs in vivo generates four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts that have been proposed to be responsible for PA-induced liver tumor formation in rats. In this present study, we determined that the same set of DHP-DNA adducts was formed upon the incubation of 7-glutathione-DHP and 7-cysteine-DHP with cultured human hepatocarcinoma HepG2 cells. These results suggest that 7-glutathione-DHP and 7-cysteine-DHP are reactive metabolites of PAs that can bind to cellular DNA to form DHP-DNA adducts in HepG2 cells, and can potentially initiate liver tumor formation.

MARSI: metabolite analogues for rational strain improvement

DEFF Research Database (Denmark)

Cardoso, João G. R.; Zeidan, Ahmad A; Jensen, Kristian

2018-01-01

reactions in an organism can be used to predict effects of MAs on cellular phenotypes. Here, we present the Metabolite Analogues for Rational Strain Improvement (MARSI) framework. MARSI provides a rational approach to strain improvement by searching for metabolites as targets instead of genes or reactions...

Metabolite identification through multiple kernel learning on fragmentation trees.

Science.gov (United States)

Shen, Huibin; Dührkop, Kai; Böcker, Sebastian; Rousu, Juho

2014-06-15

Metabolite identification from tandem mass spectrometric data is a key task in metabolomics. Various computational methods have been proposed for the identification of metabolites from tandem mass spectra. Fragmentation tree methods explore the space of possible ways in which the metabolite can fragment, and base the metabolite identification on scoring of these fragmentation trees. Machine learning methods have been used to map mass spectra to molecular fingerprints; predicted fingerprints, in turn, can be used to score candidate molecular structures. Here, we combine fragmentation tree computations with kernel-based machine learning to predict molecular fingerprints and identify molecular structures. We introduce a family of kernels capturing the similarity of fragmentation trees, and combine these kernels using recently proposed multiple kernel learning approaches. Experiments on two large reference datasets show that the new methods significantly improve molecular fingerprint prediction accuracy. These improvements result in better metabolite identification, doubling the number of metabolites ranked at the top position of the candidates list. © The Author 2014. Published by Oxford University Press.

Pectin Enhances Bio-Control Efficacy by Inducing Colonization and Secretion of Secondary Metabolites by Bacillus amyloliquefaciens SQY 162 in the Rhizosphere of Tobacco.

Directory of Open Access Journals (Sweden)

Kai Wu

Full Text Available Bacillus amyloliquefaciens is a plant-beneficial Gram-positive bacterium involved in suppressing soil-borne pathogens through the secretion of secondary metabolites and high rhizosphere competence. Biofilm formation is regarded as a prerequisite for high rhizosphere competence. In this work, we show that plant extracts affect the chemotaxis and biofilm formation of B. amyloliquefaciens SQY 162 (SQY 162. All carbohydrates tested induced the chemotaxis and biofilm formation of the SQY 162 strain; however, the bacterial growth rate was not influenced by the addition of carbohydrates. A strong chemotactic response and biofilm formation of SQY 162 were both induced by pectin through stimulation of surfactin synthesis and transcriptional expression of biofilm formation related matrix genes. These results suggested that pectin might serve as an environmental factor in the stimulation of the biofilm formation of SQY 162. Furthermore, in pot experiments the surfactin production and the population of SQY 162 in the rhizosphere significantly increased with the addition of sucrose or pectin, whereas the abundance of the bacterial pathogen Ralstonia decreased. With increased production of secondary metabolites in the rhizosphere of tobacco by SQY 162 and improved colonization density of SQY 162 in the pectin treatment, the disease incidences of bacterial wilt were efficiently suppressed. The present study revealed that certain plant extracts might serve as energy sources or environmental cues for SQY 162 to enhance the population density on tobacco root and bio-control efficacy of tobacco bacterial wilt.

Do metabolites that are produced during resistance exercise enhance muscle hypertrophy?

Science.gov (United States)

Dankel, Scott J; Mattocks, Kevin T; Jessee, Matthew B; Buckner, Samuel L; Mouser, J Grant; Loenneke, Jeremy P

2017-11-01

Many reviews conclude that metabolites play an important role with respect to muscle hypertrophy during resistance exercise, but their actual physiologic contribution remains unknown. Some have suggested that metabolites may work independently of muscle contraction, while others have suggested that metabolites may play a secondary role in their ability to augment muscle activation via inducing fatigue. Interestingly, the studies used as support for an anabolic role of metabolites use protocols that are not actually designed to test the importance of metabolites independent of muscle contraction. While there is some evidence in vitro that metabolites may induce muscle hypertrophy, the only study attempting to answer this question in humans found no added benefit of pooling metabolites within the muscle post-exercise. As load-induced muscle hypertrophy is thought to work via mechanotransduction (as opposed to being metabolically driven), it seems likely that metabolites simply augment muscle activation and cause the mechanotransduction cascade in a larger proportion of muscle fibers, thereby producing greater muscle growth. A sufficient time under tension also appears necessary, as measurable muscle growth is not observed after repeated maximal testing. Based on current evidence, it is our opinion that metabolites produced during resistance exercise do not have anabolic properties per se, but may be anabolic in their ability to augment muscle activation. Future studies are needed to compare protocols which produce similar levels of muscle activation, but differ in the magnitude of metabolites produced, or duration in which the exercised muscles are exposed to metabolites.

The hepatic metabolism of two carcinogenic dimethylbenz[c]acridines in control and induced rats: the distribution and the mutagenicity of metabolites.

Science.gov (United States)

Ye, Y; Scharping, C E; Holder, G M

1995-04-01

The major and minor metabolites of the potent polycyclic aza-aromatic carcinogens 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine, and the stereochemistry of the dihydrodiol metabolites have been previously described. The metabolite distributions produced in incubations of the aza-aromatic compounds with liver microsomes from phenobarbital- and 3-methylcholanthrene-pretreated and untreated rats, and the mutagenicity in the Ames test are described in this paper. The major metabolites of each were the alcohols produced by oxidation of the methyl group on the 8,9,10,11-ring for control and phenobarbital-induced preparations, while with 3-methylcholanthrene-induced preparations both the 7- and 9- (or 10-) monoalcohols were formed. Total monofunctionalized dihydrodiol metabolites, the 5,6- and 3,4-isomers for 7,9-dimethylbenz[c]acridine, and the 3,4-, 5,6- and 8,9-isomers for 7,10-dimethylbenz[c]acridine, constituted approximately 10% of total metabolites. As well, the K-region arene oxide was formed in substantial amounts with both compounds, accompanied in the case of 7,10-dimethylbenz[c]acridine with some 8,9-oxide. When incubations were carried out in the presence of the epoxide hydrase inhibitor 3,3,3-trichloropropane-1,2-oxide, dihydrodiol formation was almost completely inhibited and relative amounts of both phenols and oxides increased. Secondary metabolites were also formed to approximately 10% of the total products. The mutagenicity of synthetic alcohols and isolated purified metabolites was determined in the Salmonella mammalian microsome plate assay (Ames test) with strain TA100. Limited amounts of metabolites isolated precluded extensive testing, but high mutagenicities were noted for all 3,4-dihydrodiol derivatives isolated. These exceeded those of the parent aza-aromatic hydrocarbons. Alcohols were also active but less so than the parent compounds. The activation of these two dimethylbenz[c]acridines to mutagens appears to be through bay

Methodological considerations for measuring glucocorticoid metabolites in feathers

Science.gov (United States)

Berk, Sara A.; McGettrick, Julie R.; Hansen, Warren K.; Breuner, Creagh W.

2016-01-01

In recent years, researchers have begun to use corticosteroid metabolites in feathers (fCORT) as a metric of stress physiology in birds. However, there remain substantial questions about how to measure fCORT most accurately. Notably, small samples contain artificially high amounts of fCORT per millimetre of feather (the small sample artefact). Furthermore, it appears that fCORT is correlated with circulating plasma corticosterone only when levels are artificially elevated by the use of corticosterone implants. Here, we used several approaches to address current methodological issues with the measurement of fCORT. First, we verified that the small sample artefact exists across species and feather types. Second, we attempted to correct for this effect by increasing the amount of methanol relative to the amount of feather during extraction. We consistently detected more fCORT per millimetre or per milligram of feather in small samples than in large samples even when we adjusted methanol:feather concentrations. We also used high-performance liquid chromatography to identify hormone metabolites present in feathers and measured the reactivity of these metabolites against the most commonly used antibody for measuring fCORT. We verified that our antibody is mainly identifying corticosterone (CORT) in feathers, but other metabolites have significant cross-reactivity. Lastly, we measured faecal glucocorticoid metabolites in house sparrows and correlated these measurements with corticosteroid metabolites deposited in concurrently grown feathers; we found no correlation between faecal glucocorticoid metabolites and fCORT. We suggest that researchers should be cautious in their interpretation of fCORT in wild birds and should seek alternative validation methods to examine species-specific relationships between environmental challenges and fCORT. PMID:27335650

Secondary Metabolites from Higher Fungi: Discovery, Bioactivity, and Bioproduction

Science.gov (United States)

Zhong, Jian-Jiang; Xiao, Jian-Hui

Medicinal higher fungi such as Cordyceps sinensis and Ganoderma lucidum have been used as an alternative medicine remedy to promote health and longevity for people in China and other regions of the world since ancient times. Nowadays there is an increasing public interest in the secondary metabolites of those higher fungi for discovering new drugs or lead compounds. Current research in drug discovery from medicinal higher fungi involves a multifaceted approach combining mycological, biochemical, pharmacological, metabolic, biosynthetic and molecular techniques. In recent years, many new secondary metabolites from higher fungi have been isolated and are more likely to provide lead compounds for new drug discovery, which may include chemopreventive agents possessing the bioactivity of immunomodulatory, anticancer, etc. However, numerous challenges of secondary metabolites from higher fungi are encountered including bioseparation, identification, biosynthetic metabolism, and screening model issues, etc. Commercial production of secondary metabolites from medicinal mushrooms is still limited mainly due to less information about secondary metabolism and its regulation. Strategies for enhancing secondary metabolite production by medicinal mushroom fermentation include two-stage cultivation combining liquid fermentation and static culture, two-stage dissolved oxygen control, etc. Purification of bioactive secondary metabolites, such as ganoderic acids from G. lucidum, is also very important to pharmacological study and future pharmaceutical application. This review outlines typical examples of the discovery, bioactivity, and bioproduction of secondary metabolites of higher fungi origin.

Metabolism of Lutein and Zeaxanthin in Rhesus Monkeys: Identification of (3R,6′R)- and (3R,6′S)-3′-Dehydro-lutein as Common Metabolites and Comparison to Humans

Science.gov (United States)

Albert, Gesa I.; Hoeller, Ulrich; Schierle, Joseph; Neuringer, Martha; Johnson, Elizabeth J.; Schalch, Wolfgang

2012-01-01

Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3′-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 to 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3′-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6′R)-3′-dehydro-lutein and (3R,6′S)-3′-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These finding were comparable to results obtained with human plasma. PMID:18582588

Detecting Beer Intake by Unique Metabolite Patterns.

Science.gov (United States)

Gürdeniz, Gözde; Jensen, Morten Georg; Meier, Sebastian; Bech, Lene; Lund, Erik; Dragsted, Lars Ove

2016-12-02

Evaluation of the health related effects of beer intake is hampered by the lack of accurate tools for assessing intakes (biomarkers). Therefore, we identified plasma and urine metabolites associated with recent beer intake by untargeted metabolomics and established a characteristic metabolite pattern representing raw materials and beer production as a qualitative biomarker of beer intake. In a randomized, crossover, single-blinded meal study (MSt1), 18 participants were given, one at a time, four different test beverages: strong, regular, and nonalcoholic beers and a soft drink. Four participants were assigned to have two additional beers (MSt2). In addition to plasma and urine samples, test beverages, wort, and hops extract were analyzed by UPLC-QTOF. A unique metabolite pattern reflecting beer metabolome, including metabolites derived from beer raw material (i.e., N-methyl tyramine sulfate and the sum of iso-α-acids and tricyclohumols) and the production process (i.e., pyro-glutamyl proline and 2-ethyl malate), was selected to establish a compliance biomarker model for detection of beer intake based on MSt1. The model predicted the MSt2 samples collected before and up to 12 h after beer intake correctly (AUC = 1). A biomarker model including four metabolites representing both beer raw materials and production steps provided a specific and accurate tool for measurement of beer consumption.

Pharmaceutically active secondary metabolites of marine actinobacteria.

Science.gov (United States)

Manivasagan, Panchanathan; Venkatesan, Jayachandran; Sivakumar, Kannan; Kim, Se-Kwon

2014-04-01

Marine actinobacteria are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinobacteria from terrestrial sources have been studied and screened since the 1950s, for many important antibiotics, anticancer, antitumor and immunosuppressive agents. However, frequent rediscovery of the same compounds from the terrestrial actinobacteria has made them less attractive for screening programs in the recent years. At the same time, actinobacteria isolated from the marine environment have currently received considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. They are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, antitumor, cytotoxic, cytostatic, anti-inflammatory, anti-parasitic, anti-malaria, antiviral, antioxidant, anti-angiogenesis, etc. In this review, an evaluation is made on the current status of research on marine actinobacteria yielding pharmaceutically active secondary metabolites. Bioactive compounds from marine actinobacteria possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens. With the increasing advancement in science and technology, there would be a greater demand for new bioactive compounds synthesized by actinobacteria from various marine sources in future. Copyright © 2013 Elsevier GmbH. All rights reserved.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

International Nuclear Information System (INIS)

Greaves, Alana K.; Su, Guanyong; Letcher, Robert J.

2016-01-01

metabolism of OPEs was rapid, with the exception of TEP (no metabolism observed). • Relative metabolism rates (pmol/min/mg protein): TNBP > TBOEP > TPHP > TDCIPP > TCIPP. • OPE metabolite (i.e., OP diesters) formation was shown for all 5 OP triesters. • Structure-dependent metabolism rates and OP diester formation were observed.

Environmentally relevant organophosphate triesters in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a hepatic microsomal assay

Energy Technology Data Exchange (ETDEWEB)

Greaves, Alana K. [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada); Su, Guanyong, E-mail: guanyong.su85@gmail.com [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada); Letcher, Robert J., E-mail: robert.letcher@canada.ca [Wildlife and Landscape Directorate, Science and Technology Branch, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3 (Canada); Department of Chemistry, Carleton University, Ottawa, ON K1S 5B6 (Canada)

2016-10-01

microsomes. • The metabolism of OPEs was rapid, with the exception of TEP (no metabolism observed). • Relative metabolism rates (pmol/min/mg protein): TNBP > TBOEP > TPHP > TDCIPP > TCIPP. • OPE metabolite (i.e., OP diesters) formation was shown for all 5 OP triesters. • Structure-dependent metabolism rates and OP diester formation were observed.

2'-Deoxyguanosine as a surrogate trapping agent for DNA reactive drug metabolites.

Science.gov (United States)

Häkkinen, Merja R; Laine, Jaana E; Juvonen, Risto O; Auriola, Seppo; Häyrinen, Jukka; Pasanen, Markku

2011-11-10

Drug metabolism can result in the production of highly reactive metabolites that may form adducts with cellular macromolecules, and thus initiate adverse drug reactions, cause toxicity, and even require the withdrawal of drug from the market. In this study, a 2'-deoxyguanosine (dG)-based chemical trapping test system was developed for use as a fast screening tool for DNA adducting metabolites of new drug candidates. Reactive metabolites were generated from parent compounds in in vitro incubations with phenobarbital-induced mouse liver microsomes, human liver microsomes and different recombinant human CYP enzymes in the presence of dG. The formed dG-adducts were separated, characterized and their stability was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was evaluated with six test compounds, aflatoxin B1, estrone, clozapine, tolcapone, ticlopidine and imipramine. Estrone and aflatoxin B1 formed dG adducts with phenobarbital-induced mouse liver microsomes, human liver microsomes and human recombinant CYP enzymes. Adduct formation was also observed with tolcapone when phenobarbital-induced mouse liver microsomes were used as the enzyme source. The stability of each formed adduct was independent of the different enzyme sources. No dG-adducts were identified with ticlopidine, clozapine and imipramine. Compared to other classical DNA reactivity tests, e.g. Ames test, the present surrogate endpoint, the dG adduct, is faster, enables the characterization of the formed compounds, and also permits the investigation of more unstable adducts. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

Directory of Open Access Journals (Sweden)

Valerie E. Ryman

2016-01-01

Full Text Available Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1 metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE, can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE, is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

Prototype of an intertwined secondary-metabolite supercluster

Science.gov (United States)

Phillipp Wiemann; Chun-Jun. Guo; Jonathan M. Palmer; Relebohile Sekonyela; Clay C.C. Wang; Nancy P. Keller

2013-01-01

The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the...

A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids

DEFF Research Database (Denmark)

Syed, Muzeeb; Srinivas, Nuggehally R

2016-01-01

Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the an...

[Identification of saponins from Panax notoginseng in metabolites of rats].

Science.gov (United States)

Shen, Wen-Wen; Zhang, Yin; Qiu, Shou-Bei; Zhu, Fen-Xia; Jia, Xiao-Bin; Tang, Dao-Quan; Chen, Bin

2017-10-01

UPLC-QTOF-MS/MS was used to identify metabolites in rat blood, urine and feces after the administration of n-butanol extract derived from steamed notoginseng. The metabolic process of saponins came from steamed notoginseng was analyzed. The metabolites were processed by PeakView software, and identified according to the structural characteristics of prototype compounds and the accurate qualitative and quantitative changes of common metabolic pathways. Four saponins metabolites were identified based on MS/MS information of metabolites, namely ginsenoside Rh₄, Rk₃, Rk₁, Rg₅,and their 15 metabolites were verified. The metabolic pathways of the four ginsenosides in n-butanol extract included glucuronidation, desugar, sulfation, dehydromethylation, and branch loss. The metabolites of main active saponin components derived from steamed Panax notoginseng were analyzed from the perspective of qualitative analysis. And the material basis for the efficacy of steamed notoginseng was further clarified. Copyright© by the Chinese Pharmaceutical Association.

Aeration-Controlled Formation of Acid in Heterolactic Fermentations

DEFF Research Database (Denmark)

Adler-Nissen, Jens

1994-01-01

fermentation processes should be analyzed as fed-batch fermentations with oxygen as the limiting substrate. Addition of fructose in limited amounts leads to the formation of one half mole of acetic acid for each mole fructose, thus offering an alternative mechanism for controlling acetic acid formation.......Controlled aeration of Leuconostoc mesenteroides was studied as a possible mechanism for control of the formation of acetic acid, a metabolite of major influence on the taste of lactic fermented foods. Fermentations were carried out in small scale in a medium in which growth was limited...... by the buffer capacity only. Ethanol and acetic acid formed during the fermentation were analyzed by rapid head space gas chromatography, and the ratio of the molar concentrations of these two volatiles quantitatively predicted the balance between the formation of acetic acid and lactic acid. The oxygen...

A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry.

Science.gov (United States)

Rydevik, Axel; Hansson, Annelie; Hellqvist, Anna; Bondesson, Ulf; Hedeland, Mikael

2015-07-01

A new model is presented that can be used to screen for bioactivation of drugs. The evaluation of toxicity is an important step in the development of new drugs. One way to detect possible toxic metabolites is to use trapping agents such as glutathione. Often human liver microsomes are used as a metabolic model in initial studies. However, there is a need for alternatives that are easy to handle, cheap, and can produce large amounts of metabolites. In the presented study, paracetamol, mefenamic acid, and diclofenac, all known to form reactive metabolites in humans, were incubated with the fungus Cunninghamella elegans and the metabolites formed were characterized with ultra high performance liquid chromatography coupled to a quadrupole time of flight mass spectrometer. Interestingly, glutathione conjugates formed by the fungus were observed for all three drugs and their retention times and MS/MS spectra matched those obtained in a comparative experiment with human liver microsomes. These findings clearly demonstrated that the fungus is a suitable trapping model for toxic biotransformation products. Cysteine conjugates of all three test drugs were also observed with high signal intensities in the fungal incubates, giving the model a further indicator of drug bioactivation. To our knowledge, this is the first demonstration of the use of a fungal model for the formation and trapping of reactive drug metabolites. The investigated model is cheap, easy to handle, it does not involve experimental animals and it can be scaled up to produce large amounts of metabolites. Copyright © 2014 John Wiley & Sons, Ltd.

Secondary metabolites from Eremostachys laciniata

DEFF Research Database (Denmark)

Calis, Ihsan; Güvenc, Aysegül; Armagan, Metin

2008-01-01

), and forsythoside B (18), and five flavone derivatives, luteolin (19), luteolin 7-O-β-D-glucopyranoside (20), luteolin 7-O-(6''-O-β-D-apiofuranosyl)-β-D-glucopyranoside (21), apigenin 7-O-β-D-glucopyranoside (22), and apigenin 7-O-(6''-O-p-coumaroyl)-β-D-glucopyranoside (23). The structures of the metabolites were...... elucidated from spectroscopic (UV, IR, 1D- and 2D-NMR) and ESI-MS evidence, as well as from their specific optical rotation. The presence of these metabolites of three different classes strongly supports the close relationship of the genera Eremostachys and Phlomis....

Cyanobacteria as Cell Factories to Produce Plant Secondary Metabolites

OpenAIRE

Xue, Yong; He, Qingfang

2015-01-01

Cyanobacteria represent a promising platform for the production of plant secondary metabolites. Their capacity to express plant P450 proteins, which have essential functions in the biosynthesis of many plant secondary metabolites, makes cyanobacteria ideal for this purpose, and their photosynthetic capability allows cyanobacteria to grow with simple nutrient inputs. This review summarizes the advantages of using cyanobacteria to transgenically produce plant secondary metabolites. Some techniq...

Generation of ERα-floxed and knockout mice using the Cre/LoxP system

International Nuclear Information System (INIS)

Antonson, P.; Omoto, Y.; Humire, P.; Gustafsson, J.-Å.

2012-01-01

Highlights: ► ERα floxed and knockout mice were generated. ► Disruption of the ERα gene results in sterility in both male and female mice. ► ERα −/− mice have ovaries with hemorrhagic follicles and hypoplastic uterus. ► Female ERα −/− mice develop obesity. -- Abstract: Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

Bioactive secondary metabolites from chrozophora plicata

International Nuclear Information System (INIS)

Riaz, N.; Tabussum, A.; Saleem, M.; Parveen, S.

2014-01-01

Fourteen compounds (1-14) namely 3-carbomethoxyindole (1), 6-hydroxy-7-methoxycoumarin (2), methyl-4-hydroxybenzoate (3), 4-hydroxybenzoic acid (4), p-coumaric acid (5), oleanolic acid (6), 4-ketopinoresinol (7), apigenin (8), apigenin-7-p-coumerate (9) apigenin-7-O-beta-D-glucopyranoside (10), acacetin-7-O-(4-hydroxy-E-cinnamoyl-(6)-beta-D-glucopyranoside (11), apigenin-7-O-(4-hydroxy-E-cinnamoyl-(6)-beta-D-glucopyranoside (12), -bis(4-hydroxy-E-cinnamoyl))glucopyranoside 6 apigenin 7-O-beta-D-(2 (13) and apigenin 7-O-rutinoside (14) were isolated from the methanolic extract of the whole plant of Chrozophora plicata. The structures of these compounds were confirmed by UV, IR, NMR (1H and 13C) spectroscopy and mass spectrometry (EIMS, HREIMS, FABMS, HRFABMS) and in comparison with reported data in literature. These isolates (1-14) showed DPPH radical scavenging and enzyme inhibitory activities against enzyme acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and lipoxygenase (LOX). (author)

An update on organohalogen metabolites produced by basidiomycetes

NARCIS (Netherlands)

Field, J.A.; Wijnberg, J.B.P.A.

2003-01-01

Basidiomycetes are an ecologically important group of higher fungi known for their widespread capacity to produce organohalogen metabolites. To date, 100 different organohalogen metabolites (mostly chlorinated) have been identified from strains in 70 genera of Basidiomycetes. This manuscript

Metabolome analysis - mass spectrometry and microbial primary metabolites

DEFF Research Database (Denmark)

Højer-Pedersen, Jesper Juul

2008-01-01

, and therefore sample preparation is critical for metabolome analysis. The three major steps in sample preparation for metabolite analysis are sampling, extraction and concentration. These three steps were evaluated for the yeast Saccharomyces cerevisiae with primary focus on analysis of a large number...... of metabolites by one method. The results highlighted that there were discrepancies between different methods. To increase the throughput of cultivation, S. cerevisiae was grown in microtitier plates (MTPs), and the growth was found to be comparable with cultivations in shake flasks. The carbon source was either...... a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high-accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI...

New secondary metabolites of phenylbutyrate in humans and rats.

Science.gov (United States)

Kasumov, Takhar; Brunengraber, Laura L; Comte, Blandine; Puchowicz, Michelle A; Jobbins, Kathryn; Thomas, Katherine; David, France; Kinman, Renee; Wehrli, Suzanne; Dahms, William; Kerr, Douglas; Nissim, Itzhak; Brunengraber, Henri

2004-01-01

Phenylbutyrate is used to treat inborn errors of ureagenesis, malignancies, cystic fibrosis, and thalassemia. High-dose phenylbutyrate therapy results in toxicity, the mechanism of which is unexplained. The known metabolites of phenylbutyrate are phenylacetate, phenylacetylglutamine, and phenylbutyrylglutamine. These are excreted in urine, accounting for a variable fraction of the dose. We identified new metabolites of phenylbutyrate in urine of normal humans and in perfused rat livers. These metabolites result from interference between the metabolism of phenylbutyrate and that of carbohydrates and lipids. The new metabolites fall into two categories, glucuronides and phenylbutyrate beta-oxidation side products. Two questions are raised by these data. First, is the nitrogen-excreting potential of phenylbutyrate diminished by ingestion of carbohydrates or lipids? Second, does competition between the metabolism of phenylbutyrate, carbohydrates, and lipids alter the profile of phenylbutyrate metabolites? Finally, we synthesized glycerol esters of phenylbutyrate. These are partially bioavailable in rats and could be used to administer large doses of phenylbutyrate in a sodium-free, noncaustic form.

Novel urinary metabolite of d-delta-tocopherol in rats

International Nuclear Information System (INIS)

Chiku, S.; Hamamura, K.; Nakamura, T.

1984-01-01

A novel metabolite of d-delta-tocopherol was isolated from the urine of rats given d-3,4-[ 3 H 2 ]-delta-tocopherol intravenously. The metabolite was collected from the urine of rats given d-delta-tocopherol in the same manner as that of the labeled compound. It was found that the metabolites consisted of sulfate conjugates. The portion of the major metabolite released with sulfatase was determined to be 2,8-dimethyl-2-(2'-carboxyethyl)-6-chromanol by infrared spectra, nuclear magnetic resonance spectra, and mass spectra. The proposed structure was confirmed by comparing the analytical results with those of a synthetically derived compound. As a result of the structural elucidation of this novel metabolite, a pathway for the biological transformation of delta-tocopherol is proposed which is different from that of alpha-tocopherol. A characteristic feature of the pathway is the absence of any opening of the chroman ring throughout the sequence

Circulating prostacyclin metabolites in the dog

International Nuclear Information System (INIS)

Taylor, B.M.; Shebuski, R.J.; Sun, F.F.

1983-01-01

The present study was designed to determine the concentration of prostacyclin (PGI2) metabolites in the blood of the dog. After a bolus i.v. dose of [11 beta- 3 H]PGI2 (5 micrograms/kg) into each of five dogs, blood samples were withdrawn at 0.33, 0.67, 1, 3, 5, 20, 30, 60 and 120 min postdrug administration. Plasma samples were extracted and the radioactive components were analyzed by two-dimensional thin-layer chromatography with autoradiofluorography and radio-high-performance liquid chromatography. The compounds were identified by comparing their mobility with synthetic standards; only parallel responses observed in both tests constituted positive identification. Seven metabolites were identified by these two techniques: 6-keto-prostaglandin (PG)F1 alpha; 6-keto-PGE1; 2,3-dinor-6-keto-PGF 1 alpha; 2,3-dinor-13,14-dihydro-6,15-diketo-20-carboxyl PGF 1 alpha; and 2,3,18,19-tetranor-13,14-dihydro-6,15-diketo-20-carboxyl PGF 1 alpha. Several additional compounds, both polar and nonpolar in nature, which did not co-chromatograph with any of our standards were also detected. Early samples consisted predominantly of 6-keto-PGF 1 alpha and other 20-carbon metabolites. By 30 min, the predominant metabolites were the 16- and 18-carbon dicarboxylic acids. By 60 min, 85% of the radioactivity was associated with two unidentified polar compounds. The evidence suggests that 6-keto-PGF 1 alpha probably reflects only the transient levels of freshly entering PGI2 in the circulation, whereas levels of the most polar metabolites (e.g., dihydro-diketo-carboxyl tetranor-PGF 2 alpha) may be a better measure of the overall PGI2 presence due to its longer half-life in circulation

Pharmacokinetics of ifosfamide and some metabolites in children

NARCIS (Netherlands)

Kaijser, G. P.; de Kraker, J.; Bult, A.; Underberg, W. J.; Beijnen, J. H.

1998-01-01

The pharmacokinetics of ifosfamide and some metabolites in children was investigated. The patients received various doses of ifosfamide, mostly by continuous infusion, over several days. The penetration of ifosfamide and its metabolites into the cerebrospinal fluid was also studied in four cases.

Production of cyathane type secondary metabolites by submerged cultures of Hericium erinaceus and evaluation of their antibacterial activity by direct bioautography.

Science.gov (United States)

Shen, T; Morlock, G; Zorn, H

2015-01-01

Fungi of the phylum Basidiomycota are well-known to form a broad spectrum of biologically active secondary metabolites, especially low molecular weight compounds such as terpenoids. Hericium erinaceus produces various cyathane type diterpenoids including erinacines. However, no quantitative data and production kinetics have been reported on the biosynthesis of the erinacines C and P in submerged cultures. In the present study, the production of erinacine C was optimized, and the product formation kinetics as well as the antimicrobial activity were studied by high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC) and direct bioautography. Oatmeal and Edamin ® K were identified to be crucial media components for an efficient production of erinacine C. The highest concentrations of erinacine C were obtained in the optimized culture medium on the 9 th culture day (approximately 260 mg L -1 ). The production of erinacine P was strongly time dependent. The maximum concentration of erinacine P of 184 mg L -1 was observed on the third culture day. Afterwards, the concentrations of erinacine P decreased while the concentrations of erinacine C steadily increased. Comparable results were obtained by HPTLC with UV detection and HPLC with diode-array detection (DAD) analyses. Direct bioautography allowed for an additional analysis of the antimicrobial activity of the secondary metabolites. The C and N sources oatmeal and Edamin ® K induced the formation of erinacine C. Detailed product formation kinetics of the erinacines C and P have been reported for the first time. HPTLC combined with the Aliivibrio fischeri bioassay allowed for an instant detection of cyathane diterpenoids in crude extracts and for an evaluation of the antimicrobial activity of the secondary metabolites directly on the plate.

Development of a general-purpose method for cell purification using Cre/loxP-mediated recombination.

Science.gov (United States)

Kuroki, Shunsuke; Akiyoshi, Mika; Ideguchi, Ko; Kitano, Satsuki; Miyachi, Hitoshi; Hirose, Michiko; Mise, Nathan; Abe, Kuniya; Ogura, Atsuo; Tachibana, Makoto

2015-06-01

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general-purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock-in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP-mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1-Cre-transgenic (tg) embryos almost equally as efficiently as from Nr5a1-hCD271-tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh-Cre-tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. © 2015 Wiley Periodicals, Inc.

Novel pyrazine metabolites found in polymyxin biosynthesis by Paenibacillus polymyxa

DEFF Research Database (Denmark)

Beck, Hans Christian; Hansen, Anne M; Lauritsen, Frants R

2003-01-01

A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2,5-diisoprop......A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2...

Assessing the associations of blood metabolites with osteoporosis: a Mendelian randomization study.

Science.gov (United States)

Liu, Li; Wen, Yan; Zhang, Lei; Xu, Peng; Liang, Xiao; Du, Yanan; Li, Ping; He, Awen; Fan, QianRui; Hao, Jingcan; Wang, Wenyu; Guo, Xiong; Shen, Hui; Tian, Qing; Zhang, Feng; Deng, Hong-Wen

2018-03-01

Osteoporosis is a metabolic bone disease. The impact of blood metabolites on the development of osteoporosis remains elusive now. To explore the relationship between blood metabolites and osteoporosis. We used 2,286 unrelated Caucasian subjects as discovery samples and 3,143 unrelated Caucasian subjects from the Framingham heart study (FHS) as replication samples. Bone mineral density (BMD) were measured using dual-energy X-ray absorptiometry. Genome-wide SNP genotyping was performed using Affymetrix Human SNP Array 6.0 (for discovery samples) and Affymetrix SNP 500K and 50K array (for FHS replication samples). The SNP sets significantly associated with blood metabolites were obtained from a published whole-genome sequencing study. For each subject, the genetic risk score (GRS) of metabolite was calculated from the genotype data of metabolite associated SNP sets. Pearson correlation analysis was conducted to evaluate the potential impact of blood metabolites on the variations bone phenotypes. 10,000 permutations were conducted to calculate the empirical P value and false discovery rate (FDR). 481 blood metabolites were analyzed in this study. We identified multiple blood metabolites associated with hip BMD, such as 1,5-anhydroglucitol(1,5-AG) (Pdiscovery metabolites on the variations of BMD, and identified several candidate blood metabolites for osteoporosis.

Metabolites of alectinib in human: their identification and pharmacological activity

Directory of Open Access Journals (Sweden)

Mika Sato-Nakai

2017-07-01

Full Text Available Two metabolites (M4 and M1b in plasma and four metabolites (M4, M6, M1a and M1b in faeces were detected through the human ADME study following a single oral administration of [14C]alectinib, a small-molecule anaplastic lymphoma kinase inhibitor, to healthy subjects. In the present study, M1a and M1b, which chemical structures had not been identified prior to the human ADME study, were identified as isomers of a carboxylate metabolite oxidatively cleaved at the morpholine ring. In faeces, M4 and M1b were the main metabolites, which shows that the biotransformation to M4 and M1b represents two main metabolic pathways for alectinib. In plasma, M4 was a major metabolite and M1b was a minor metabolite. The contribution to in vivo pharmacological activity of these circulating metabolites was assessed from their in vitro pharmacological activity and plasma protein binding. M4 had a similar cancer cell growth inhibitory activity and plasma protein binding to that of alectinib, suggesting its contribution to the antitumor activity of alectinib, whereas the pharmacological activity of M1b was insignificant.

Metabolomics and Cheminformatics Analysis of Antifungal Function of Plant Metabolites.

Science.gov (United States)

Cuperlovic-Culf, Miroslava; Rajagopalan, NandhaKishore; Tulpan, Dan; Loewen, Michele C

2016-09-30

Fusarium head blight (FHB), primarily caused by Fusarium graminearum , is a devastating disease of wheat. Partial resistance to FHB of several wheat cultivars includes specific metabolic responses to inoculation. Previously published studies have determined major metabolic changes induced by pathogens in resistant and susceptible plants. Functionality of the majority of these metabolites in resistance remains unknown. In this work we have made a compilation of all metabolites determined as selectively accumulated following FHB inoculation in resistant plants. Characteristics, as well as possible functions and targets of these metabolites, are investigated using cheminformatics approaches with focus on the likelihood of these metabolites acting as drug-like molecules against fungal pathogens. Results of computational analyses of binding properties of several representative metabolites to homology models of fungal proteins are presented. Theoretical analysis highlights the possibility for strong inhibitory activity of several metabolites against some major proteins in Fusarium graminearum , such as carbonic anhydrases and cytochrome P450s. Activity of several of these compounds has been experimentally confirmed in fungal growth inhibition assays. Analysis of anti-fungal properties of plant metabolites can lead to the development of more resistant wheat varieties while showing novel application of cheminformatics approaches in the analysis of plant/pathogen interactions.

Metabolite Profiling of Candidatus Liberibacter Infection in Hamlin Sweet Oranges.

Science.gov (United States)

Hung, Wei-Lun; Wang, Yu

2018-04-18

Huanglongbing (HLB), also known as citrus greening disease, caused by Candidatus Liberibacter asiaticus (CLas), is considered the most serious citrus disease in the world. CLas infection has been shown to greatly affect metabolite profiles in citrus fruits. However, because of uneven distribution of CLas throughout the tree and a minimum bacterial titer requirement for polymerase chain reaction (PCR) detection, the infected trees may test false negative. To prevent this, metabolites of healthy Hamlin oranges (CLas-) obtained from the citrus undercover protection systems (CUPS) were investigated. Comparison of the metabolite profile of juice obtained from CLas- and CLas+ (asymptomatic and symptomatic) trees revealed significant differences in both volatile and nonvolatile metabolites. However, no consistent pattern could be observed in alcohols, esters, sesquiterpenes, sugars, flavanones, and limonoids as compared to previous studies. These results suggest that CLas may affect metabolite profiles of citrus fruits earlier than detecting infection by PCR. Citric acid, nobiletin, malic acid, and phenylalanine were identified as the metabolic biomarkers associated with the progression of HLB. Thus, the differential metabolites found in this study may serve as the biomarkers of HLB in its early stage, and the metabolite signature of CLas infection may provide useful information for developing a potential treatment strategy.

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage▿

OpenAIRE

Broberg, Anders; Jacobsson, Karin; Ström, Katrin; Schnürer, Johan

2007-01-01

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydro...

Aspergillus flavus secondary metabolites: more than just aflatoxins

Science.gov (United States)

Aspergillus flavus is best known for producing the family of potent carcinogenic secondary metabolites known as aflatoxins. However, this opportunistic plant and animal pathogen also produces numerous other secondary metabolites, many of which have also been shown to be toxic. While about forty of t...

Direct detection of glucuronide metabolites of lidocaine in sheep urine.

Science.gov (United States)

Doran, Gregory S; Smith, Alistair K; Rothwell, Jim T; Edwards, Scott H

2018-02-15

The anaesthetic lidocaine is metabolised quickly to produce a series of metabolites, including several hydroxylated metabolites, which are further metabolised by addition of a glucuronic acid moiety. Analysis of these glucuronide metabolites in urine is performed indirectly by cleaving the glucuronic acid group using β-glucuronidase. However, direct analysis of intact glucuronide conjugates is a more straightforward approach as it negates the need for long hydrolysis incubations, and minimises the oxidation of sensitive hydrolysis products, while also distinguishing between the two forms of hydroxylated metabolites. A method was developed to identify three intact glucuronides of lidocaine in sheep urine using LC-MS/MS, which was further confirmed by the synthesis of glucuronide derivatives of 3OH-MEGX and 4OH-LIDO. Direct analysis of urine allowed the detection of the glucuronide metabolites of hydroxylidocaine (OH-LIDO), hydroxyl-monoethylglycinexylidide (OH-MEGX), and hydroxy-2,6-xylidine (OH-XYL). Analysis of urine before and after β-glucuronidase digestion showed that the efficiency of hydrolysis of these glucuronide metabolites may be underestimated in some studies. Analysis of urine in the current study from three different sheep with similar glucuronide metabolite concentrations resulted in different hydrolysis efficiencies, which may have been a result of different levels of substrate binding by matrix components, preventing enzyme cleavage. The use of direct analysis of intact glucuronides has the benefit of being less influenced by these matrix effects, while also allowing analysis of unstable metabolites like 4OH-XYL, which rapidly oxidises after hydrolysis. Additionally, direct analysis is less expensive and less time consuming, while providing more information about the status of hydroxylated metabolites in urine. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Metabolite characterization in serum samples from normal healthy ...

African Journals Online (AJOL)

Metabolite characterization in serum samples from normal healthy human subjects by 1H and 13C NMR spectroscopy. D Misra, U Bajpai. Abstract. One and two dimensional NMR spectroscopy has been employed to characterize the various metabolites of serum control healthy samples. Two dimensional heteronuclear ...

UV-guided isolation of fungal metabolites by HSCCC

DEFF Research Database (Denmark)

Dalsgaard, P.W.; Nielsen, K.F.; Larsen, Thomas Ostenfeld

2005-01-01

Analytical standardised reversed phase liquid chromatography (RPLC) data can be helpful in finding a suitable solvent combination for isolation of fungal metabolites by high-speed counter current chromatography. Analysis of the distribution coefficient (K-D) of fungal metabolites in a series...... peptides from a crude fungal extract....

Effect of metabolites produced by Trichoderma species against ...

African Journals Online (AJOL)

Metabolites released from Trichoderma viride, T. polysporum, T. hamatum and T. aureoviride were tested in culture medium against Ceratocystis paradoxa, which causes black seed rot in oil palm sprouted seeds. The Trichoderma metabolites had similar fungistatic effects on the growth of C. paradoxa except those from T.

Reparation and Immunomodulating Properties of Bacillus sp. Metabolites from Permafrost.

Science.gov (United States)

Kalenova, L F; Melnikov, V P; Besedin, I M; Bazhin, A S; Gabdulin, M A; Kolyvanova, S S

2017-09-01

An ointment containing metabolites of Bacillus sp. microorganisms isolated from permafrost samples was applied onto the skin wound of BALB/c mice. Metabolites isolated during culturing of Bacillus sp. at 37°C produced a potent therapeutic effect and promoted wound epithelialization by 30% in comparison with the control (ointment base) and by 20% in comparison with Solcoseryl. Treatment with Bacillus sp. metabolites stimulated predominantly humoral immunity, reduced the time of wound contraction and the volume of scar tissue, and promoted complete hair recovery. These metabolites can be considered as modulators of the wound process with predominance of regeneration mechanisms.

Extracellular Metabolites from Industrial Microalgae and Their Biotechnological Potential

Directory of Open Access Journals (Sweden)

Lu Liu

2016-10-01

Full Text Available Industrial microalgae, as a big family of promising producers of renewable biomass feedstock, have been commercially exploited for functional food, living feed and feed additives, high-value chemicals in nutraceuticals, cosmeceuticals, and chemical reagents. Recently, microalgae have also been considered as a group that might play an important role in biofuel development and environmental protection. Almost all current products of industrial microalgae are derived from their biomass; however, large amounts of spent cell-free media are available from mass cultivation that is mostly unexploited. In this contribution we discuss that these media, which may contain a remarkable diversity of bioactive substances are worthy to be recovered for further use. Obviously, the extracellular metabolites from industrial microalgae have long been neglected in the development of production methods for valuable metabolites. With the advances in the last ten years, more and more structures and properties from extracellular metabolites have been identified, and the potential utilization over wide fields is attracting attention. Some of these extracellular metabolites can be potentially used as drugs, antioxidants, growth regulators or metal chelators. The purpose of this review is to provide an overview of the known extracellular metabolites from industrial microalgae which might be of commercial interest. The attention mainly focuses on the reports of extracellular bioactive metabolites and their potential application in biotechnology.

Metabolites from inhalation of aerosolized S-8 synthetic jet fuel in rats.

Science.gov (United States)

Tremblay, Raphael T; Martin, Sheppard A; Fisher, Jeffrey W

2011-01-01

Alternative fuels are being considered for civilian and military uses. One of these is S-8, a replacement jet fuel synthesized using the Fischer-Tropsch process, which contains no aromatic compounds and is mainly composed of straight and branched alkanes. Metabolites of S-8 fuel in laboratory animals have not been identified. The goal of this study was to identify metabolic products from exposure to aerosolized S-8 and a designed straight-chain alkane/polyaromatic mixture (decane, undecane, dodecane, tridecane, tetradecane, pentadecane, naphthalene, and 2-methylnaphthalene) in male Fischer 344 rats. Collected blood and tissue samples were analyzed for 70 straight and branched alcohols and ketones ranging from 7 to 15 carbons. No fuel metabolites were observed in the blood, lungs, brain, and fat following S-8 exposure. Metabolites were detected in the liver, urine, and feces. Most of the metabolites were 2- and 3-position alcohols and ketones of prominent hydrocarbons with very few 1- or 4-position metabolites. Following exposure to the alkane mixture, metabolites were observed in the blood, liver, and lungs. Interestingly, heavy metabolites (3-tridecanone, 2-tridecanol, and 2-tetradecanol) were observed only in the lung tissues possibly indicating that metabolism occurred in the lungs. With the exception of these heavy metabolites, the metabolic profiles observed in this study are consistent with previous studies reporting on the metabolism of individual alkanes. Further work is needed to determine the potential metabolic interactions of parent, primary, and secondary metabolites and identify more polar metabolites. Some metabolites may have potential use as biomarkers of exposure to fuels.

Formation and degradation of ethylenethiourea (ETU) in soil and water under tropical conditions

NARCIS (Netherlands)

Ruiz-Suárez, L.E.; Geissen, V.; Jarquin Sánchez, A.; Castro Chan, R.A.; Bello-Mendoza, R.

2013-01-01

Mancozeb is a fungicide frequently used in tropical countries. It rapidly decomposes into ethylenethiourea (ETU), a more stable and toxic metabolite than mancozeb that is, therefore, regarded as a pollutant of concern. The objective was to study ETU formation and decay kinetics in soil and water

Metabolite production by species of Stemphylium

DEFF Research Database (Denmark)

Olsen, Kresten Jon Kromphardt; Rossman, Amy; Andersen, Birgitte

2018-01-01

metabolites were found to be important for distinguishing species, while some unknown metabolites were also found to have important roles in distinguishing species of Stemphylium. This study is the first of its kind to investigate the chemical potential of Stemphylium across the whole genus.......Morphology and phylogeny has been used to distinguish members of the plant pathogenic fungal genus Stemphylium. A third method for distinguishing species is by chemotaxonomy. The main goal of the present study was to investigate the chemical potential of Stemphylium via HPLC-UV-MS analysis, while...

Trajectory optimization for A S.S.T.O. using in-flight LOX collection

Science.gov (United States)

Saint-Mard, M.; Hendrick, P.

A key point for a space mission (launch of a satellite, earth observation,…) is the optimization of the vehicle trajectory in order to burn the smallest quantity of propelant and then maximize the payload. This is true for evay space vehicle, but especially it is a crucial point for a Single-Stage-To-Orbit (SSTO) where the choice of a bad trajectory can result in an unrealizable vehicle due to the large airbreathing part of the flight In this study, we discuss the trajectory optimization for a Vertical Take-Off and Horizontal Landing (VTOHL) SSTO using supersonic in-flight atmospheric oxygen collection during a cruise phase (constant speed & constant altitude). This collected oxygen is stored in the LOX tanks and reused in the final rocket phase. This SSTO bas a Blended Body aerodynamic configuration as the one chosen by Lockheed Martin for its new space launcher (VentureStar and X-33). This SSTO uses rocket engines from take-off to Mach 1.7 and also for the exoatmospheric flight phase (that means for an altitude higher than 30km and a Mach number evolution from 6.8 to about 20). Between these two rocket phases, the SSTO is propelled by a subsonic ramjet. To perform this study, we use 2 computer programs (running on a home Computer): the first one allows to estimate the SSTO performances (TOGW, dry weight, hydrogen and oxygen consumptions) for a fixed payload mass and the second one permits the evaluation of the payload mass for a fixed TOGW.

Northrop Grumman TR202 LOX/LH2 Deep Throttling Engine Technology Project Status

Science.gov (United States)

Gromski, Jason; Majamaki, Annik; Chianese, Silvio; Weinstock, Vladimir; Kim, Tony S.

2010-01-01

NASA's Propulsion and Cryogenic Advanced Development (PCAD) project is currently developing enabling propulsion technologies in support of future lander missions. To meet lander requirements, several technical challenges need to be overcome, one of which is the ability for the descent engine(s) to operate over a deep throttle range with cryogenic propellants. To address this need, PCAD has enlisted Northrop Grumman Aerospace Systems (NGAS) in a technology development effort associated with the TR202 engine. The TR202 is a LOX/LH2 expander cycle engine driven by independent turbopump assemblies and featuring a variable area pintle injector similar to the injector used on the TR200 Apollo Lunar Module Descent Engine (LMDE). Since the Apollo missions, NGAS has continued to mature deep throttling pintle injector technology. The TR202 program has completed two series of pintle injector testing. The first series of testing used ablative thrust chambers and demonstrated igniter operation as well as stable performance at discrete points throughout the designed 10:1 throttle range. The second series was conducted with calorimeter chambers and demonstrated injector performance at discrete points throughout the throttle range as well as chamber heat flow adequate to power an expander cycle design across the throttle range. This paper provides an overview of the TR202 program, describing the different phases and key milestones. It describes how test data was correlated to the engine conceptual design. The test data obtained has created a valuable database for deep throttling cryogenic pintle technology, a technology that is readily scalable in thrust level.

Identification of Unique Metabolites of the Designer Opioid Furanyl Fentanyl.

Science.gov (United States)

Goggin, Melissa M; Nguyen, An; Janis, Gregory C

2017-06-01

The illicit drug market has seen an increase in designer opioids, including fentanyl and methadone analogs, and other structurally unrelated opioid agonists. The designer opioid, furanyl fentanyl, is one of many fentanyl analogs clandestinely synthesized for recreational use and contributing to the fentanyl and opioid crisis. A method has been developed and validated for the analysis of furanyl fentanyl and furanyl norfentanyl in urine specimens from pain management programs. Approximately 10% of samples from a set of 500 presumptive heroin-positive urine specimens were found to contain furanyl fentanyl, with an average concentration of 33.8 ng/mL, and ranging from 0.26 to 390 ng/mL. Little to no furanyl norfentanyl was observed; therefore, the furanyl fentanyl specimens were further analyzed by untargeted high-resolution mass spectrometry to identify other metabolites. Multiple metabolites, including a dihydrodiol metabolite, 4-anilino-N-phenethyl-piperidine (4-ANPP) and a sulfate metabolite were identified. The aim of the presented study was to identify the major metabolite(s) of furanyl fentanyl and estimate their concentrations for the purpose of toxicological monitoring. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Loss of metabolites from monkey striatum during PET with FDOPA

DEFF Research Database (Denmark)

Cumming, P; Munk, O L; Doudet, D

2001-01-01

diffusion of [(18)F]fluorodopamine metabolites from brain. Consequently, time-radioactivity recordings of striatum are progressively influenced by metabolite loss. In linear analyses, the net blood-brain clearance of FDOPA (K(D)(i), ml g(-1) min(-1)) can be corrected for this loss by the elimination rate...... constant k(Lin)(cl) (min(-1)). Similarly, the DOPA decarboxylation rate constant (k(D)(3), min(-1)) calculated by compartmental analysis can also be corrected for metabolite loss by the elimination rate constant k(DA)(9) (min(-1)). To compare the two methods, we calculated the two elimination rate...... of the estimate was substantially improved upon correction for metabolite loss. The rate constants for metabolite loss were higher in MPTP-lesioned monkey striatum than in normal striatum. The high correlation between individual estimates of k(Lin)(cl) and k(DA)(9) suggests that both rate constants reveal loss...

Identification of a Classical Mutant in the Industrial Host Aspergillus niger by Systems Genetics: LaeA Is Required for Citric Acid Production and Regulates the Formation of Some Secondary Metabolites

DEFF Research Database (Denmark)

Niu, Jing; Arentshorst, Mark; Nair, P. Deepa S.

2015-01-01

could provide new insights into the transcriptional control mechanisms related to citric acid production in A. niger. Interestingly, the secondary metabolite profile of a ΔlaeA strain differed from the wild-type strain, showing both decreased and increased metabolite levels, indicating that LaeA is also...

Role of oxidative metabolites of cocaine in toxicity and addiction: oxidative stress and electron transfer.

Science.gov (United States)

Kovacic, Peter

2005-01-01

Cocaine is one of the principal drugs of abuse. Although impressive advances have been made, unanswered questions remain concerning mechanism of toxicity and addiction. Discussion of action mode usually centers on receptor binding and enzyme inhibition, with limited attention to events at the molecular level. This review provides extensive evidence in support of the hypothesis that oxidative metabolites play important roles comprising oxidative stress (OS), reactive oxygen species (ROS), and electron transfer (ET). The metabolites include norcocaine and norcocaine derivatives: nitroxide radical, N-hydroxy, nitrosonium, plus cocaine iminium and formaldehyde. Observed formation of ROS is rationalized by redox cycling involving several possible ET agents. Three potential ones are present in the form of oxidative metabolites, namely, nitroxide, nitrosonium, and iminium. Most attention has been devoted to the nitroxide-hydroxylamine couple which has been designated by various investigators as the principal source of ROS. The proximate ester substituent is deemed important for intramolecular stabilization of reactive intermediates. Reduction potential of nitroxide is in accord with plausibility of ET in the biological milieu. Toxicity by cocaine, with evidence for participation of OS, is demonstrated for many body components, including liver, central nervous system, cardiovascular system, reproductive system, kidney, mitochondria, urine, and immune system. Other adverse effects associated with ROS comprise teratogenesis and apoptosis. Examples of ROS generated are lipid peroxides and hydroxyl radical. Often observed were depletion of antioxidant defenses, and protection by added antioxidants, such as, thiol, salicylate, and deferoxamine. Considerable evidence supports the contention that oxidative ET metabolites of cocaine are responsible for much of the observed OS. Quite significantly, the pro-oxidant, toxic effects, including generation of superoxide and lipid peroxyl

Identification of Volatile Secondary Metabolites from an Endophytic Microfungus Aspergillus Nomius KUB105

International Nuclear Information System (INIS)

Lateef Adebola Azeez; Lateef Adebola Azeez; Sepiah Muid; Bolhassan Mohamad Hasnul

2016-01-01

Microfungi are a highly diverse group of micro-organisms and important components of the ecosystem with great potential for diverse metabolite production. During a survey of microfungi on leaves in a National Park in Sarawak, an uncommon endophytic microfungus Aspergillus nomius was encountered. The metabolite production of this microfungus was investigated by growing it in a liquid basal medium for 2 weeks. Gas Chromatography - Mass Spectrometry (GC-MS) and Fourier Transform Infrared (FTIR) profiling of the secondary metabolites produced by this microfungus in the liquid medium revealed the presence of 46 different secondary metabolites. The metabolites include saturated hydrocarbons, alkyl halides, alcohols and an unsaturated hydrocarbon. Majority of the metabolites produced were saturated hydrocarbons. Tetracosane, Icosane and 10-Methylicosane were the most abundant metabolites identified while heptadecane and 2,4-dimethylundecane were the least abundant respectively. This study is the first GC-MS and FTIR report of secondary metabolites from A. nomius. The results from this study confirm the ability of microfungi to produce diverse metabolites, including saturated hydrocarbons. (author)

Thermodynamics-based Metabolite Sensitivity Analysis in metabolic networks.

Science.gov (United States)

Kiparissides, A; Hatzimanikatis, V

2017-01-01

The increasing availability of large metabolomics datasets enhances the need for computational methodologies that can organize the data in a way that can lead to the inference of meaningful relationships. Knowledge of the metabolic state of a cell and how it responds to various stimuli and extracellular conditions can offer significant insight in the regulatory functions and how to manipulate them. Constraint based methods, such as Flux Balance Analysis (FBA) and Thermodynamics-based flux analysis (TFA), are commonly used to estimate the flow of metabolites through genome-wide metabolic networks, making it possible to identify the ranges of flux values that are consistent with the studied physiological and thermodynamic conditions. However, unless key intracellular fluxes and metabolite concentrations are known, constraint-based models lead to underdetermined problem formulations. This lack of information propagates as uncertainty in the estimation of fluxes and basic reaction properties such as the determination of reaction directionalities. Therefore, knowledge of which metabolites, if measured, would contribute the most to reducing this uncertainty can significantly improve our ability to define the internal state of the cell. In the present work we combine constraint based modeling, Design of Experiments (DoE) and Global Sensitivity Analysis (GSA) into the Thermodynamics-based Metabolite Sensitivity Analysis (TMSA) method. TMSA ranks metabolites comprising a metabolic network based on their ability to constrain the gamut of possible solutions to a limited, thermodynamically consistent set of internal states. TMSA is modular and can be applied to a single reaction, a metabolic pathway or an entire metabolic network. This is, to our knowledge, the first attempt to use metabolic modeling in order to provide a significance ranking of metabolites to guide experimental measurements. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier

Leach and mold resistance of essential oil metabolites

Science.gov (United States)

Carol A. Clausen; Vina W. Yang

2011-01-01

Purified primary metabolites from essential oils were previously shown to be bioactive inhibitors of mold fungi on unleached Southern pine sapwood, either alone or in synergy with a second metabolite. This study evaluated the leachability of these compounds in Southern pine that was either dip- or vacuum-treated. Following laboratory leach tests, specimens were...

Possible endocrine disrupting effects of parabens and their metabolites

DEFF Research Database (Denmark)

Boberg, Julie; Taxvig, Camilla; Christiansen, Sofie

2010-01-01

Parabens are preservatives used in a wide range of cosmetic products, including products for children, and some are permitted in foods. However, there is concern for endocrine disrupting effects. This paper critically discusses the conclusions of recent reviews and original research papers...... and provides an overview of studies on toxicokinetics. After dermal uptake, parabens are hydrolyzed and conjugated and excreted in urine. Despite high total dermal uptake of paraben and metabolites,little intact paraben can be recovered in blood and urine. Paraben metabolites may play a role in the endocrine...... disruption seen in experimental animals and studies are needed to determine human levels of parabens and metabolites. Overall, the estrogenic burden of parabens and their metabolites in blood may exceed the action of endogenous estradiol in childhood and the safety margin for propylparaben is very low when...

Urinary metabolites of tetrahydronorharman in the rat

Energy Technology Data Exchange (ETDEWEB)

Greiner, B.; Rommelspacher, H.

1982-01-01

The metabolism of THN in the rat was studied in vivo by use of /sup 14/C-radiolabelled compound. Structures of major urinary metabolites were determined by exact spectral data. Their concentrations were measured by liquid scintillation counting. It was found that THN is submitted to endogenous transformation, and that the excreted derivatives form three groups of similar concentration: unchanged substance, hydroxylated/conjugated compounds, and aromatic metabolites. Structures and proposed pathways are summed in diagram.

Urinary metabolites of tetrahydronorharman in the rat

International Nuclear Information System (INIS)

Greiner, B.; Rommelspacher, H.

1982-01-01

The metabolism of THN in the rat was studied in vivo by use of 14 C-radiolabelled compound. Structures of major urinary metabolites were determined by exact spectral data. Their concentrations were measured by liquid scintillation counting. It was found that THN is submitted to endogenous transformation, and that the excreted derivatives form three groups of similar concentration: unchanged substance, hydroxylated/conjugated compounds, and aromatic metabolites. Structures and proposed pathways are summed in diagram

A latex metabolite benefits plant fitness under root herbivore attack

OpenAIRE

Huber, M.; Epping, J.; Gronover, C.S.; Fricke, J.; Aziz, Z.; Brillatz, T.; Swyers, M.; Köllner, T.G.; Vogel, H.; Hammerbacher, A.; Triebwasser-Freese, D.; Robert, C.A.M.; Verhoeven, K.; Preite, V.; Gershenzon, J.

2016-01-01

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major n...

Effect of Acinetobacter sp on metalaxyl degradation and metabolite profile of potato seedlings (Solanum tuberosum L. alpha variety.

Directory of Open Access Journals (Sweden)

Fabiola G Zuno-Floriano

Full Text Available One of the most serious diseases in potato cultivars is caused by the pathogen Phytophthora infestans, which affects leaves, stems and tubers. Metalaxyl is a fungicide that protects potato plants from Phytophthora infestans. In Mexico, farmers apply metalaxyl 35 times during the cycle of potato production and the last application is typically 15 days before harvest. There are no records related to the presence of metalaxyl in potato tubers in Mexico. In the present study, we evaluated the effect of Acinetobacter sp on metalaxyl degradation in potato seedlings. The effect of bacteria and metalaxyl on the growth of potato seedlings was also evaluated. A metabolite profile analysis was conducted to determine potential molecular biomarkers produced by potato seedlings in the presence of Acinetobacter sp and metalaxyl. Metalaxyl did not affect the growth of potato seedlings. However, Acinetobacter sp strongly affected the growth of inoculated seedlings, as confirmed by plant length and plant fresh weights which were lower in inoculated potato seedlings (40% and 27%, respectively compared to the controls. Acinetobacter sp also affected root formation. Inoculated potato seedlings showed a decrease in root formation compared to the controls. LC-MS/MS analysis of metalaxyl residues in potato seedlings suggests that Acinetobacter sp did not degrade metalaxyl. GC-TOF-MS platform was used in metabolic profiling studies. Statistical data analysis and metabolic pathway analysis allowed suggesting the alteration of metabolic pathways by both Acinetobacter sp infection and metalaxyl treatment. Several hundred metabolites were detected, 137 metabolites were identified and 15 metabolic markers were suggested based on statistical change significance found with PLS-DA analysis. These results are important for better understanding the interactions of putative endophytic bacteria and pesticides on plants and their possible effects on plant metabolism.

Human lunar mission capabilities using SSTO, ISRU and LOX-augmented NTR technologies: A preliminary assessment

Science.gov (United States)

Borowski, Stanley K.

1995-10-01

The feasibility of conducting human missions to the Moon is examined assuming the use of three 'high leverage' technologies: (1) a single-stage-to-orbit (SSTO) launch vehicle, (2) 'in-situ' resource utilization (ISRU)--specifically 'lunar-derived' liquid oxygen (LUNOX), and (3) LOX-augmented nuclear thermal rocket (LANTR) propulsion. Lunar transportation system elements consisting of a LANTR-powered lunar transfer vehicle (LTV) and a chemical propulsion lunar landing/Earth return vehicle (LERV) are configured to fit within the 'compact' dimensions of the SSTO cargo bay (diameter: 4.6 m/length: 9.0 m) while satisfying an initial mass in low Earth orbit (IMLEO) limit of approximately 60 t (3 SSTO launches). Using approximately 8 t of LUNOX to 'reoxidize' the LERV for a 'direct return' flight to Earth reduces its size and mass allowing delivery to LEO on a single 20 t SSTO launch. Similarly, the LANTR engine's ability to operate at any oxygen/ hydrogen mixture ratio from 0 to 7 with high specific impulse (approximately 940 to 515 s) is exploited to reduce hydrogen tank volume, thereby improving packaging of the LANTR LTV's 'propulsion' and 'propellant modules'. Expendable and reusable, piloted and cargo missions and vehicle designs are presented along with estimates of LUNOX production required to support the different mission modes. Concluding remarks address the issue of lunar transportation system costs from the launch vehicle perspective.

Differences in metabolite profiles caused by pre-analytical blood processing procedures.

Science.gov (United States)

Nishiumi, Shin; Suzuki, Makoto; Kobayashi, Takashi; Yoshida, Masaru

2018-05-01

Recently, the use of metabolomic analysis of human serum and plasma for biomarker discovery and disease diagnosis in clinical studies has been increasing. The feasibility of using a metabolite biomarker for disease diagnosis is strongly dependent on the metabolite's stability during pre-analytical blood processing procedures, such as serum or plasma sampling and sample storage prior to centrifugation. However, the influence of blood processing procedures on the stability of metabolites has not been fully characterized. In the present study, we compared the levels of metabolites in matched human serum and plasma samples using gas chromatography coupled with mass spectrometry and liquid chromatography coupled with mass spectrometry. In addition, we evaluated the changes in plasma metabolite levels induced by storage at room temperature or at a cold temperature prior to centrifugation. As a result, it was found that 76 metabolites exhibited significant differences between their serum and plasma levels. Furthermore, the pre-centrifugation storage conditions significantly affected the plasma levels of 45 metabolites. These results highlight the importance of blood processing procedures during metabolome analysis, which should be considered during biomarker discovery and the subsequent use of biomarkers for disease diagnosis. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis.

Science.gov (United States)

Tsikas, Dimitrios; Trettin, Arne; Zörner, Alexander A; Gutzki, Frank-Mathias

2011-05-15

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions

Enantioselective biotransformation of propranolol to the active metabolite 4-hydroxypropranolol by endophytic fungi

Directory of Open Access Journals (Sweden)

Keyller Bastos Borges

2011-01-01

Full Text Available The enantioselective biotransformation of propranolol (Prop by the endophytic fungi Phomopsis sp., Glomerella cingulata, Penicillium crustosum, Chaetomium globosum and Aspergillus fumigatus was investigated by studying the kinetics of the aromatic hydroxylation reaction with the formation of 4-hydroxypropranolol (4-OH-Prop. Both Prop enantiomers were consumed by the fungi in the biotransformation process, but the 4-hydroxylation reaction yielded preferentially (--(S-4-OH-Prop. The quantity of metabolites biosynthesized varied slightly among the evaluated endophytic fungi. These results show that all investigated endophytic fungi could be used as biosynthetic tools in biotransformation processes to obtain the enantiomers of 4-OH-Prop.

Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

International Nuclear Information System (INIS)

Oosthuizen, M.J.; Engels, F.; Van Esch, B.; Henricks, P.A.; Nijkamp, F.P.

1990-01-01

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed

10 CFR 26.163 - Cutoff levels for drugs and drug metabolites.

Science.gov (United States)

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Cutoff levels for drugs and drug metabolites. 26.163... the Department of Health and Human Services § 26.163 Cutoff levels for drugs and drug metabolites. (a... testing of specimens to determine whether they are negative for the indicated drugs and drug metabolites...

Options for human ``return to the moon'' using tomorrow's SSTO, ISRU, and LOX-augmented NTR technologies

Science.gov (United States)

Borowski, Stanley K.

1996-03-01

The feasibility of conducting human missions to the Moon is examined assuming the use of three ``high leverage'' technologies: (1) a single-stage-to-orbit (SSTO) launch vehicle, (2) ``in-situ'' resource utilization (ISRU)—specifically ``lunar-derived'' liquid oxygen (LUNOX), and (3) LOX-augmented nuclear thermal rocket (LANTR) propulsion. Lunar transportation system elements consisting of a LANTR-powered lunar transfer vehicle (LTV) and a chemical propulsion lunar landing/Earth return vehicle (LERV) are configured to fit within the ``compact'' dimensions of the SSTO cargo bay (diameter: 4.6 m/length: 9.0 m) while satisfying an initial mass in low Earth orbit (IMLEO) limit of ˜60 t (3 SSTO launches). Using ˜8 t of LUNOX to ``reoxidize'' the LERV for a ``direct return'' flight to Earth reduces its size and mass allowing delivery to LEO on a single 20 t SSTO launch. Similarly, the LANTR engine's ability to operate at any oxygen/hydrogen mixture ratio from 0 to 7 with high specific impulse (˜940 to 515 s) is exploited to reduce hydrogen tank volume, thereby improving packaging of the LANTR LTV's ``propulsion'' and ``propellant modules''. Expendable and reusable, piloted and cargo missions and vehicle designs are presented along with estimates of LUNOX production required to support the different mission modes.

Metabolite variability in Caribbean sponges of the genus Aplysina

Directory of Open Access Journals (Sweden)

Monica Puyana

Full Text Available Abstract Sponges of the genus Aplysina are among the most common benthic animals on reefs of the Caribbean, and display a wide diversity of morphologies and colors. Tissues of these sponges lack mineralized skeletal elements, but contain a dense spongin skeleton and an elaborate series of tyrosine-derived brominated alkaloid metabolites that function as chemical defenses against predatory fishes, but do not deter some molluscs. Among the earliest marine natural products to be isolated and identified, these metabolites remain the subject of intense interest for commercial applications because of their activities in various bioassays. In this study, crude organic extracts from 253 sponges from ten morphotypes among the species Aplysina archeri,Aplysina bathyphila,Aplysina cauliformis,Aplysina fistularis,Aplysina fulva,A. insularis, and Aplysina lacunosa were analyzed by liquid chromatography–mass spectrometry (LC–MS to characterize the pattern of intra- and interspecific variabilities of the twelve major secondary metabolites present therein. Patterns across Aplysina species ranged from the presence of mostly a single compound, fistularin-3, in A. cauliformis, to a mixture of metabolites present in the other species. These patterns did not support the biotransformation hypothesis for conversion of large molecular weight molecules to smaller ones for the purpose of enhanced defense. Discriminant analyses of the metabolite data revealed strong taxonomic patterns that support a close relationship between A. fistularis,A. fulva and A. insularis, while two morphotypes of A. cauliformis (lilac creeping vs. brown erect were very distinct. Two morphotypes of A. lacunosa, one with hard tissue consistency, the other soft and thought to belong to a separate genus (Suberea, had very similar chemical profiles. Of the twelve metabolites found among samples, variation in fistularin-3, dideoxyfistularin-3 and hydroxyaerothionin provided the most predictive

Classification and modelling of non-extractable residue (NER) formation of xenobiotics in soil - a synthesis

DEFF Research Database (Denmark)

Kästner, M.; Nowak, K. M.; Miltner, A.

2014-01-01

This review provides a comprehensive overview about NER formation and attempts to classify the various types. Xenobiotic NER derived from parent pesticides (or other environmental contaminants) and primary metabolites sorbed or entrapped within the soil organic matter (type I) or covalently bound...

MSD-MAP: A Network-Based Systems Biology Platform for Predicting Disease-Metabolite Links.

Science.gov (United States)

Wathieu, Henri; Issa, Naiem T; Mohandoss, Manisha; Byers, Stephen W; Dakshanamurthy, Sivanesan

2017-01-01

Cancer-associated metabolites result from cell-wide mechanisms of dysregulation. The field of metabolomics has sought to identify these aberrant metabolites as disease biomarkers, clues to understanding disease mechanisms, or even as therapeutic agents. This study was undertaken to reliably predict metabolites associated with colorectal, esophageal, and prostate cancers. Metabolite and disease biological action networks were compared in a computational platform called MSD-MAP (Multi Scale Disease-Metabolite Association Platform). Using differential gene expression analysis with patient-based RNAseq data from The Cancer Genome Atlas, genes up- or down-regulated in cancer compared to normal tissue were identified. Relational databases were used to map biological entities including pathways, functions, and interacting proteins, to those differential disease genes. Similar relational maps were built for metabolites, stemming from known and in silico predicted metabolite-protein associations. The hypergeometric test was used to find statistically significant relationships between disease and metabolite biological signatures at each tier, and metabolites were assessed for multi-scale association with each cancer. Metabolite networks were also directly associated with various other diseases using a disease functional perturbation database. Our platform recapitulated metabolite-disease links that have been empirically verified in the scientific literature, with network-based mapping of jointly-associated biological activity also matching known disease mechanisms. This was true for colorectal, esophageal, and prostate cancers, using metabolite action networks stemming from both predicted and known functional protein associations. By employing systems biology concepts, MSD-MAP reliably predicted known cancermetabolite links, and may serve as a predictive tool to streamline conventional metabolomic profiling methodologies. Copyright© Bentham Science Publishers; For any

Novel pyrazine metabolites found in polymyxin biosynthesis by Paenibacillus polymyxa

DEFF Research Database (Denmark)

Beck, Hans Christian; Hansen, Anne M; Lauritsen, Frants R

2003-01-01

A complex mixture of methyl-branched alkyl-substituted pyrazines was found in the growth medium of the polymyxin-producing bacterium Paenibacillus polymyxa, and of these, seven are new natural compounds. A total of 19 pyrazine metabolites were identified. The dominant metabolite was 2...... supplementation. The other pyrazine metabolites, all related pyrazines with either one, two or three alkyl substituents, were identified by means of their mass spectral data and/or co-elution with authentic standards....

A combined genetic and multi medium approach revels new secondary metabolites in Aspergillus nidulans

DEFF Research Database (Denmark)

Klejnstrup, Marie Louise; Nielsen, Morten Thrane; Frisvad, Jens Christian

Secondary metabolites are a diverse group of metabolites which serve as important natural sources of drugs for treating diseases. The availability of full genome sequences of several filamentous fungi has revealed a large genetic potential for production of secondary metabolites that are not obse......Secondary metabolites are a diverse group of metabolites which serve as important natural sources of drugs for treating diseases. The availability of full genome sequences of several filamentous fungi has revealed a large genetic potential for production of secondary metabolites...... that are not observed under standard laboratory conditions. Genetic approaches have proven a fruitfull strategy towards the production and identification of these unknown metabolites. Examples include deletion of the cclA1 and laeA2 genes in A. nidulans which affects the expression of secondary metabolites including...... monodictyphenone and terrequinone A respectively. We have deleted the cclA gene in A. nidulans and grown the mutants on several complex media to provoke the production of secondary metabolites. This resulted in the production of several metabolites not previously reported from A. nidulans. Some of these have been...

Antibacterial Secondary Metabolites from the Cave Sponge Xestospongia sp.

Directory of Open Access Journals (Sweden)

Sridevi Ankisetty

2012-05-01

Full Text Available Chemical investigation of the cave sponge Xestospongia sp. resulted in the isolation of three new polyacetylenic long chain compounds along with two known metabolites. The structures of the new metabolites were established by NMR and MS analyses. The antibacterial activity of the new metabolites was also evaluated.

Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils.

Science.gov (United States)

Docherty, J C; Wilson, T W

1987-10-29

Arachidonic acid metabolism in human neutrophils stimulated in vitro with the calcium ionophore A23187 was studied using combined HPLC and radioimmunoassays. Indomethacin (0.1 and 1.0 microM) caused a 300% increase in LTB4 formation in neutrophils stimulated with A23187. 5-, 12- and 15-HETE levels were also increased. In the presence of exogenous arachidonic acid 1.0 microM Indomethacin caused a 37% increase in LTB4 formation. Acetyl Salicylic Acid and Ibuprofen had no effect on the formation of lipoxygenase metabolites. The effect of indomethacin on LTB4 formation does not appear to be due to a simple redirection of substrate arachidonic acid from the cyclooxygenase to the lipoxygenase pathways.

Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

Science.gov (United States)

García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

2016-02-01

MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human plasma using LC-MS/MS.

Science.gov (United States)

Allard, Marie; Khoudour, Nihel; Rousseau, Benoît; Joly, Charlotte; Costentin, Charlotte; Blanchet, Benoît; Tournigand, Christophe; Hulin, Anne

2017-08-05

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of regorafenib (REGO), its two metabolites regorafenib-M2 and regorafenib-M5, sorafenib (SORA), and its N-oxide metabolite in human plasma. Separation is achieved on an Hypersil Gold ® column using a gradient elution of 10mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3mL/min. After addition of two internal standards and a protein precipitation, the supernatant is diluted two-fold in a 0.1% (v/v) formic acid solution. Two selected reaction monitoring transitions are used, for each analyte, one for quantitation and the second one for confirmation. The standard curves are ranged from 50 to 5 000ng/mL for REGO and its metabolites and 80 to 5 000ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of the analytes under various conditions. The method is usually used in clinical practice in order to improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for hepatocellular carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Potential of small-molecule fungal metabolites in antiviral chemotherapy.

Science.gov (United States)

Roy, Biswajit G

2017-08-01

Various viral diseases, such as acquired immunodeficiency syndrome, influenza, and hepatitis, have emerged as leading causes of human death worldwide. Scientific endeavor since invention of DNA-dependent RNA polymerase of pox virus in 1967 resulted in better understanding of virus replication and development of various novel therapeutic strategies. Despite considerable advancement in every facet of drug discovery process, development of commercially viable, safe, and effective drugs for these viruses still remains a big challenge. Decades of intense research yielded a handful of natural and synthetic therapeutic options. But emergence of new viruses and drug-resistant viral strains had made new drug development process a never-ending battle. Small-molecule fungal metabolites due to their vast diversity, stereochemical complexity, and preapproved biocompatibility always remain an attractive source for new drug discovery. Though, exploration of therapeutic importance of fungal metabolites has started early with discovery of penicillin, recent prediction asserted that only a small percentage (5-10%) of fungal species have been identified and much less have been scientifically investigated. Therefore, exploration of new fungal metabolites, their bioassay, and subsequent mechanistic study bears huge importance in new drug discovery endeavors. Though no fungal metabolites so far approved for antiviral treatment, many of these exhibited high potential against various viral diseases. This review comprehensively discussed about antiviral activities of fungal metabolites of diverse origin against some important viral diseases. This also highlighted the mechanistic details of inhibition of viral replication along with structure-activity relationship of some common and important classes of fungal metabolites.

Global Prioritization of Disease Candidate Metabolites Based on a Multi-omics Composite Network

Science.gov (United States)

Yao, Qianlan; Xu, Yanjun; Yang, Haixiu; Shang, Desi; Zhang, Chunlong; Zhang, Yunpeng; Sun, Zeguo; Shi, Xinrui; Feng, Li; Han, Junwei; Su, Fei; Li, Chunquan; Li, Xia

2015-01-01

The identification of disease-related metabolites is important for a better understanding of metabolite pathological processes in order to improve human medicine. Metabolites, which are the terminal products of cellular regulatory process, can be affected by multi-omic processes. In this work, we propose a powerful method, MetPriCNet, to predict and prioritize disease candidate metabolites based on integrated multi-omics information. MetPriCNet prioritized candidate metabolites based on their global distance similarity with seed nodes in a composite network, which integrated multi-omics information from the genome, phenome, metabolome and interactome. After performing cross-validation on 87 phenotypes with a total of 602 metabolites, MetPriCNet achieved a high AUC value of up to 0.918. We also assessed the performance of MetPriCNet on 18 disease classes and found that 4 disease classes achieved an AUC value over 0.95. Notably, MetPriCNet can also predict disease metabolites without known disease metabolite knowledge. Some new high-risk metabolites of breast cancer were predicted, although there is a lack of known disease metabolite information. A predicted disease metabolic landscape was constructed and analyzed based on the results of MetPriCNet for 87 phenotypes to help us understand the genetic and metabolic mechanism of disease from a global view. PMID:26598063

10 CFR 26.133 - Cutoff levels for drugs and drug metabolites.

Science.gov (United States)

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Cutoff levels for drugs and drug metabolites. 26.133... § 26.133 Cutoff levels for drugs and drug metabolites. Subject to the provisions of § 26.31(d)(3)(iii), licensees and other entities may specify more stringent cutoff levels for drugs and drug metabolites than...

Polar metabolites of polycyclic aromatic compounds from fungi are potential soil and groundwater contaminants

DEFF Research Database (Denmark)

Boll, Esther Sørensen; Johnsen, Anders R.; Christensen, Jan H.

2015-01-01

and either hydroxylated or oxidized to carboxylic acids at the methyl group. The metabolism of the sulfur-containing heterocyclic PAC resulted in sulfate conjugates. The sorption of the PAC metabolites to three soils was determined using a batch equilibrium method, and partition coefficients (Kd's) were......-methylphenanthrene, 1-methylpyrene), and one sulfur-containing heterocyclic PAC (dibenzothiophene). Fifty-eight metabolites were tentatively identified; metabolites from the un-substituted PACs were hydroxylated and sulfate conjugated, whereas metabolites from alkyl-substituted PACs were sulfate conjugated...... calculated for fourteen representative metabolites. Sulfate conjugated metabolites displayed Kd's below 70 whereas the metabolites with both a sulfate and a carboxylic acid group had Kd's below 2.8. The low Kd's of water-soluble PAC metabolites indicate high mobility in soil and a potential for leaching...

Co-evolution of secondary metabolite gene clusters and their host

DEFF Research Database (Denmark)

Kjærbølling, Inge; Vesth, Tammi Camilla; Frisvad, Jens Christian

Secondary metabolite gene cluster evolution is mainly driven by two events: gene duplication and annexation and horizontal gene transfer. Here we use comparative genomics of Aspergillus species to investigate the evolution of secondary metabolite (SM) gene clusters across a wide spectrum of speci....... We investigate the dynamic evolutionary relationship between the cluster and the host by examining the genes within the cluster and the number of homologous genes found within the host and in closely related species.......Secondary metabolite gene cluster evolution is mainly driven by two events: gene duplication and annexation and horizontal gene transfer. Here we use comparative genomics of Aspergillus species to investigate the evolution of secondary metabolite (SM) gene clusters across a wide spectrum of species...

Biodegradation of clofibric acid and identification of its metabolites

International Nuclear Information System (INIS)

Salgado, R.; Oehmen, A.; Carvalho, G.; Noronha, J.P.; Reis, M.A.M.

2012-01-01

Graphical abstract: Metabolites produced during clofibric acid biodegradation. Highlights: ► Clofibric acid is biodegradable. ► Mainly heterotrophic bacteria degraded the clofibric acid. ► Metabolites of clofibric acid biodegradation were identified. ► The metabolic pathway of clofibric acid biodegradation is proposed. - Abstract: Clofibric acid (CLF) is the pharmaceutically active metabolite of lipid regulators clofibrate, etofibrate and etofyllinclofibrate, and it is considered both environmentally persistent and refractory. This work studied the biotransformation of CLF in aerobic sequencing batch reactors (SBRs) with mixed microbial cultures, monitoring the efficiency of biotransformation of CLF and the production of metabolites. The maximum removal achieved was 51% biodegradation (initial CLF concentration = 2 mg L −1 ), where adsorption and abiotic removal mechanisms were shown to be negligible, showing that CLF is indeed biodegradable. Tests showed that the observed CLF biodegradation was mainly carried out by heterotrophic bacteria. Three main metabolites were identified, including α-hydroxyisobutyric acid, lactic acid and 4-chlorophenol. The latter is known to exhibit higher toxicity than the parent compound, but it did not accumulate in the SBRs. α-Hydroxyisobutyric acid and lactic acid accumulated for a period, where nitrite accumulation may have been responsible for inhibiting their degradation. A metabolic pathway for the biodegradation of CLF is proposed in this study.

Metabolites: messengers between the microbiota and the immune system.

Science.gov (United States)

Levy, Maayan; Thaiss, Christoph A; Elinav, Eran

2016-07-15

The mammalian intestine harbors one of the largest microbial densities on Earth, necessitating the implementation of control mechanisms by which the host evaluates the state of microbial colonization and reacts to deviations from homeostasis. While microbial recognition by the innate immune system has been firmly established as an efficient means by which the host evaluates microbial presence, recent work has uncovered a central role for bacterial metabolites in the orchestration of the host immune response. In this review, we highlight examples of how microbiota-modulated metabolites control the development, differentiation, and activity of the immune system and classify them into functional categories that illustrate the spectrum of ways by which microbial metabolites influence host physiology. A comprehensive understanding of how microbiota-derived metabolites shape the human immune system is critical for the rational design of therapies for microbiota-driven diseases. © 2016 Levy et al.; Published by Cold Spring Harbor Laboratory Press.

Filling and mining the reactive metabolite target protein database.

Science.gov (United States)

Hanzlik, Robert P; Fang, Jianwen; Koen, Yakov M

2009-04-15

The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible (http://tpdb.medchem.ku.edu:8080/protein_database/) resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of "-omics" studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites.

Relationship between measurements of blood oxidative metabolites and skin reaction in irradiated rats

International Nuclear Information System (INIS)

Kaneko, Takashi; Goto, Jun; Nomiya, Takuma; Nemoto, Kenji

2011-01-01

Recently, oxidative metabolites have been able to be measured by simple small device. It has been reported that the value of oxidative metabolites increases under several conditions such as hypertension, smoking, diabetes mellitus, etc. Radiation used in radiotherapy also causes free radicals and oxidative metabolites, and irradiation causes dermatitis and sometimes causes skin ulcer in the irradiated site. We analyzed the relationships between the value of oxidative metabolites and skin reactions. A certain doses of radiation were irradiated to the right thigh of rats, and oxidative metabolites of rat's blood from caudal vein were measured by d-reactive oxygen metabolites (ROMs) test using an exclusive device. Skin reactions were evaluated according to a skin-reaction grading system from the day before irradiation to day 38 after irradiation. As a results, a significant correlation was shown between irradiation dose and skin grade. And a significant correlation was also shown between the value of oxidative metabolites and irradiation dose. The increase in oxidative metabolites was seen in the Day 16 after irradiation, and that corresponded with the appearance of skin reaction. It was suggested that the value of oxidative metabolites seems to be useful for estimating degree of skin reaction and time to appear skin reaction after irradiation. (author)

Temporally-controlled site-specific recombination in zebrafish.

Directory of Open Access Journals (Sweden)

Stefan Hans

Full Text Available Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM or its active metabolite, 4-hydroxy-tamoxifen (4-OHT. Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

Lichen secondary metabolites affect growth of Physcomitrella patens by allelopathy.

Science.gov (United States)

Goga, Michal; Antreich, Sebastian J; Bačkor, Martin; Weckwerth, Wolfram; Lang, Ingeborg

2017-05-01

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.

Natural metabolites for parasitic weed management.

Science.gov (United States)

Vurro, Maurizio; Boari, Angela; Evidente, Antonio; Andolfi, Anna; Zermane, Nadjia

2009-05-01

Compounds of natural origin, such as phytotoxins produced by fungi or natural amino acids, could be used in parasitic weed management strategies by interfering with the early growth stages of the parasites. These metabolites could inhibit seed germination or germ tube elongation, so preventing attachment to the host plant, or, conversely, stimulate seed germination in the absence of the host, contributing to a reduction in the parasite seed bank. Some of the fungal metabolites assayed were very active even at very low concentrations, such as some macrocyclic trichothecenes, which at 0.1 microM strongly suppressed the germination of Orobanche ramosa L. seeds. Interesting results were also obtained with some novel toxins, such as phyllostictine A, highly active in reducing germ tube elongation and seed germination both of O. ramosa and of Cuscuta campestris Yuncker. Among the amino acids tested, methionine and arginine were particularly interesting, as they were able to suppress seed germination at concentrations lower than 1 mM. Some of the fungal metabolites tested were also able to stimulate the germination of O. ramosa seeds. The major findings in this research field are described and discussed.

Human metabolites of brevetoxin PbTx-2: Identification and confirmation of structure

Science.gov (United States)

Guo, Fujiang; An, Tianying; Rein, Kathleen S.

2010-01-01

Four metabolites were identified upon incubation of brevetoxin (PbTx-2) with human liver microsomes. Chemical transformation of PbTx-2 confirmed the structures of three known metabolites BTX-B5, PbTx-9 and 41, 43-dihydro-BTX-B5 and a previously unknown metabolite, 41, 43-dihydro-PbTx-2. These metabolites were also observed upon incubation of PbTx-2 with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Cytochrome P450 3A4 produced oxidized metabolites while other CYPs generated the reduced products. PMID:20600229

Stabilization of beta-catenin induces pancreas tumor formation.

Science.gov (United States)

Heiser, Patrick W; Cano, David A; Landsman, Limor; Kim, Grace E; Kench, James G; Klimstra, David S; Taketo, Maketo M; Biankin, Andrew V; Hebrok, Matthias

2008-10-01

beta-Catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of beta-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is among the leading causes of cancer death. Whereas activating mutations within beta-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of beta-catenin signaling in pancreas tumorigenesis. Using Cre/lox technology, we conditionally activated beta-catenin in a subset of murine pancreatic cells in vivo. Activation of beta-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based on morphologic and immunohistochemical comparisons. Interestingly, stabilization of beta-catenin blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to PDA. Instead, mice in which beta-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. These results demonstrate that activation of beta-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor.

Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

Science.gov (United States)

Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

2005-06-21

Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

Metabolite ratios as potential biomarkers for type 2 diabetes : a DIRECT study

NARCIS (Netherlands)

Molnos, Sophie; Wahl, Simone; Haid, Mark; Eekhoff, E Marelise W; Pool, René; Floegel, Anna; Deelen, Joris; Much, Daniela; Prehn, Cornelia; Breier, Michaela; Draisma, Harmen H; van Leeuwen, Nienke; Simonis-Bik, Annemarie M C; Jonsson, Anna; Willemsen, Gonneke; Bernigau, Wolfgang; Wang-Sattler, Rui; Suhre, Karsten; Peters, Annette; Thorand, Barbara; Herder, Christian; Rathmann, Wolfgang; Roden, Michael; Gieger, Christian; Kramer, Mark H H; van Heemst, Diana; Pedersen, Helle K; Gudmundsdottir, Valborg; Schulze, Matthias B; Pischon, Tobias; de Geus, Eco J C; Boeing, Heiner; Boomsma, Dorret I; Ziegler, Anette G; Slagboom, P. Eline; Hummel, Sandra; Beekman, Marian; Grallert, Harald; Brunak, Søren; McCarthy, Mark I; Gupta, Ramneek; Pearson, Ewan R; Adamski, Jerzy; 't Hart, Leen M

2018-01-01

AIMS/HYPOTHESIS: Circulating metabolites have been shown to reflect metabolic changes during the development of type 2 diabetes. In this study we examined the association of metabolite levels and pairwise metabolite ratios with insulin responses after glucose, glucagon-like peptide-1 (GLP-1) and

Structure of Pigment Metabolic Pathways and Their Contributions to White Tepal Color Formation of Chinese Narcissus tazetta var. chinensis cv Jinzhanyintai.

Science.gov (United States)

Ren, Yujun; Yang, Jingwen; Lu, Bingguo; Jiang, Yaping; Chen, Haiyang; Hong, Yuwei; Wu, Binghua; Miao, Ying

2017-09-08

Chinese narcissus ( Narcissus tazetta var. chinensis ) is one of the ten traditional flowers in China and a famous bulb flower in the world flower market. However, only white color tepals are formed in mature flowers of the cultivated varieties, which constrains their applicable occasions. Unfortunately, for lack of genome information of narcissus species, the explanation of tepal color formation of Chinese narcissus is still not clear. Concerning no genome information, the application of transcriptome profile to dissect biological phenomena in plants was reported to be effective. As known, pigments are metabolites of related metabolic pathways, which dominantly decide flower color. In this study, transcriptome profile and pigment metabolite analysis methods were used in the most widely cultivated Chinese narcissus "Jinzhanyintai" to discover the structure of pigment metabolic pathways and their contributions to white tepal color formation during flower development and pigmentation processes. By using comparative KEGG pathway enrichment analysis, three pathways related to flavonoid, carotenoid and chlorophyll pigment metabolism showed significant variations. The structure of flavonoids metabolic pathway was depicted, but, due to the lack of F3'5'H gene; the decreased expression of C4H , CHS and ANS genes; and the high expression of FLS gene, the effect of this pathway to synthesize functional anthocyanins in tepals was weak. Similarly, the expression of DXS , MCT and PSY genes in carotenoids synthesis sub-pathway was decreased, while CCD1 / CCD4 genes in carotenoids degradation sub-pathway was increased; therefore, the effect of carotenoids metabolic pathway to synthesize adequate color pigments in tepals is restricted. Interestingly, genes in chlorophyll synthesis sub-pathway displayed uniform down-regulated expression, while genes in heme formation and chlorophyll breakdown sub-pathways displayed up-regulated expression, which also indicates negative regulation

Phthalate metabolites in the European eel (Anguilla anguilla) from Mediterranean coastal lagoons.

Science.gov (United States)

Fourgous, C; Chevreuil, M; Alliot, F; Amilhat, E; Faliex, E; Paris-Palacios, S; Teil, M J; Goutte, A

2016-11-01

The levels and fate of phthalate metabolites have been poorly evaluated in fish, despite their potential ecotoxicological impacts. The present study aims to characterize the levels of phthalate metabolites in muscle tissue of yellow eels (Anguilla anguilla) from two coastal Mediterranean lagoons, during three sampling periods. Nine phthalate metabolites were detected in >70% of the samples. Slightly higher levels of phthalate metabolites were detected in March and June compared to October, suggesting possible seasonal variations in environmental release and/or phthalate metabolization process by eels. The large sample size (N=117) made it possible to explore correlations between phthalate metabolites' levels and individual parameters, such as body length, age, body condition and hepatic histo-pathologies. Body length and estimated age poorly correlated with phthalate metabolites, suggesting that eels did not accumulate phthalates during growth, contrary to persistent compounds. Eels presented different grades of hepatic fibrosis and lipidosis. A negative correlation was found between the severity of these pathologies in the liver and the sum of phthalate metabolites levels, supporting the hypothesis that eels with damaged liver are less able to metabolize xenobiotics. Copyright © 2016 Elsevier B.V. All rights reserved.

The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D

DEFF Research Database (Denmark)

Hagemann-Jensen, Michael Henrik; Uhlenbrock, Franziska Katharina; Kehlet, Stephanie

2014-01-01

For decades Selenium (Se) research has been focused on the identification of active metabolites, which are crucial for Se chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include...... ligands. A balanced cell-surface expression of NKG2D ligands is considered as an innate barrier against tumor development. Our work therefore indicates that the application of selenium compounds, which are metabolized to CH3SeH, could improve NKG2D-based immune therapy.......: Increased formation of reactive oxygen species (ROS), induction of DNA damage, triggering of apoptosis and the inhibition of angiogenesis. Here, we revealed that CH3SeH modulates cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways, which occur...

Emerging technologies, recent developments, and novel applications for drug metabolite identification.

Science.gov (United States)

Lu, Wenjie; Xu, Youzhi; Zhao, Yinglan; Cen, Xiaobo

2014-01-01

Drug metabolite identification and metabolic characteristics analysis play a crucial role in new drug research and development, because they can lead to varied efficacy, severe adverse reactions, and even toxicity. Classical methodologies for metabolite identification have mainly been based on mass spectrometry (MS) coupled with gas chromatography (GC) or liquid chromatography (LC), and some other techniques are used as complementary approaches, such as nuclear magnetic resonance (NMR). Over the past decade, more and more newly emerging techniques or technologies have been applied to metabolite identification, and are making the procedure easier and more robust, such as LC-NMR-MS, ion mobility MS, ambient ionization techniques, and imaging MS. A novel application of drug metabolite identification based on "omics" known as pharmacometabonomics is discussed, which is an interdisciplinary field that combines pre-dose metabolite profiling and chemometrics methods for data analysis and modeling, aiming to predict the responses of individuals to drugs.

In vivo MRS metabolite quantification using genetic optimization

Science.gov (United States)

Papakostas, G. A.; Karras, D. A.; Mertzios, B. G.; van Ormondt, D.; Graveron-Demilly, D.

2011-11-01

The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure.

Intact penetratin metabolite permeates across Caco-2 monolayers

DEFF Research Database (Denmark)

Birch, Ditlev; Christensen, Malene Vinther; Stærk, Dan

. Previous studies have demonstrated that cell-penetrating peptides (CPPs) may be used as carriers in order to improve the bioavailability of a therapeutic cargo like insulin after oral administration. Penetratin, a commonly used CPP, has been shown to increase the uptake of insulin across Caco-2 cell......-2 cells cultured on permeable filter inserts and in cell lysates, respectively. The epithelial permeation of penetratin and the formed metabolites was assessed by using Caco-2 monolayers cultured on permeable filter inserts. Results Preliminary data revealed that at least one specific metabolite...... is formed upon both intracellular and extracellular degradation of penetratin (figure 1A). Following incubation with epithelium for 4 hours, the metabolite permeated the Caco-2 monolayer and the concentration increased approximately 10-fold when compared to a sample collected following 15 minutes...

In vivo MRS metabolite quantification using genetic optimization

International Nuclear Information System (INIS)

Papakostas, G A; Mertzios, B G; Karras, D A; Van Ormondt, D; Graveron-Demilly, D

2011-01-01

The in vivo quantification of metabolites' concentrations, revealed in magnetic resonance spectroscopy (MRS) spectra, constitutes the main subject under investigation in this work. Significant contributions based on artificial intelligence tools, such as neural networks (NNs), with good results have been presented lately but have shown several drawbacks, regarding their quantification accuracy under difficult conditions. A general framework that encounters the quantification procedure as an optimization problem, which is solved using a genetic algorithm (GA), is proposed in this paper. Two different lineshape models are examined, while two GA configurations are applied on artificial data. Moreover, the introduced quantification technique deals with metabolite peaks' overlapping, a considerably difficult situation occurring under real conditions. Appropriate experiments have proved the efficiency of the introduced methodology, in artificial MRS data, by establishing it as a generic metabolite quantification procedure

[Effective productions of plant secondary metabolites having antitumor activity by plant cell and tissue cultures].

Science.gov (United States)

Taniguchi, Shoko

2005-06-01

Methods for the effective production of plant secondary metabolites with antitumor activity using plant cell and tissue cultures were developed. The factors in tannin productivity were investigated using culture strains producing different types of hydrolyzable tannins, i.e., gallotannins (mixture of galloylglucoses), ellagi-, and dehydroellagitannins. Production of ellagi- and dehydroellagitannins was affected by the concentrations and ratio of nitrogen sources in the medium. The formation of oligomeric ellagitannins in shoots of Oenothera tetraptera was correlated with the differentiation of tissues. Cultured cells of Eriobotrya japonica producing ursane- and oleanane-type triterpenes with antitumor activities were also established.

Application of quantitative time-lapse imaging (QTLI) for evaluation of Mrp2-based drug–drug interaction induced by liver metabolites

International Nuclear Information System (INIS)

Nakanishi, Takeo; Ikenaga, Miho; Fukuda, Hajime; Matsunaga, Norikazu; Tamai, Ikumi

2012-01-01

We previously reported a quantitative time-lapse imaging (QTLI)-based analysis method to assess drug–drug interactions (DDI) at multidrug resistance-associated protein 2 (Mrp2) in rat sandwich-cultured hepatocyte (SCH) system, utilizing the fluorescent Mrp2 substrate, 5-(and 6)-carboxy-2′,7′-dichlorofluorescein (CDF). Here, we aimed to examine the feasibility of using QTLI to evaluate DDI involving drug metabolite(s) generated in hepatocytes. We used estradiol (E2) and bilirubin as model compounds; both are not substrates of MRP2, whereas their hepatic metabolites, estradiol-17β-glucuronide (E17G) or bilirubin glucuronides, are known to be its substrates as well as inhibitors. When rat SCHs were pre-exposed with E2, fluorescence of CDF accumulated in bile canaliculi decreased depending upon both the duration of pre-exposure and the concentration of extracellular E2. The decrease corresponded with the increase in intracellular concentration of E17G in hepatocytes. Furthermore, cytotoxicity of vinblastine, a substrate of MRP2, was enhanced in SCHs treated with E2. Similarly, CDF accumulated in bile canaliculi was significantly reduced in rat SCHs pre-exposed with bilirubin. In conclusion, these results suggest that phase II biotransformation of a competitor is reflected in alteration of MRP2-mediated CDF transport detected in QTLI. The QTLI might provide a convenient platform to evaluate transporter-based DDIs involving hepatic metabolites of drug candidates without the need to identify the metabolites. -- Highlights: ► Mrp2-mediated CDF transport is inhibited by E2, but not E17G in vesicle study. ► Both E2 and E17G do not compromise CDF formation from CDFDA in hepatocytes. ► CDF accumulation in bile canaliculi is inhibited by E2 or E17G in QTLI. ► Increasing exposure to E2 decreases CDF accumulation in bile canaliculi in QTLI. ► QTLI is feasible to assess Mrp2-based DDI involving drug metabolite in hepatocytes.

Application of quantitative time-lapse imaging (QTLI) for evaluation of Mrp2-based drug–drug interaction induced by liver metabolites

Energy Technology Data Exchange (ETDEWEB)

Nakanishi, Takeo; Ikenaga, Miho; Fukuda, Hajime; Matsunaga, Norikazu; Tamai, Ikumi, E-mail: tamai@p.kanazawa-w.ac.jp

2012-09-01

We previously reported a quantitative time-lapse imaging (QTLI)-based analysis method to assess drug–drug interactions (DDI) at multidrug resistance-associated protein 2 (Mrp2) in rat sandwich-cultured hepatocyte (SCH) system, utilizing the fluorescent Mrp2 substrate, 5-(and 6)-carboxy-2′,7′-dichlorofluorescein (CDF). Here, we aimed to examine the feasibility of using QTLI to evaluate DDI involving drug metabolite(s) generated in hepatocytes. We used estradiol (E2) and bilirubin as model compounds; both are not substrates of MRP2, whereas their hepatic metabolites, estradiol-17β-glucuronide (E17G) or bilirubin glucuronides, are known to be its substrates as well as inhibitors. When rat SCHs were pre-exposed with E2, fluorescence of CDF accumulated in bile canaliculi decreased depending upon both the duration of pre-exposure and the concentration of extracellular E2. The decrease corresponded with the increase in intracellular concentration of E17G in hepatocytes. Furthermore, cytotoxicity of vinblastine, a substrate of MRP2, was enhanced in SCHs treated with E2. Similarly, CDF accumulated in bile canaliculi was significantly reduced in rat SCHs pre-exposed with bilirubin. In conclusion, these results suggest that phase II biotransformation of a competitor is reflected in alteration of MRP2-mediated CDF transport detected in QTLI. The QTLI might provide a convenient platform to evaluate transporter-based DDIs involving hepatic metabolites of drug candidates without the need to identify the metabolites. -- Highlights: ► Mrp2-mediated CDF transport is inhibited by E2, but not E17G in vesicle study. ► Both E2 and E17G do not compromise CDF formation from CDFDA in hepatocytes. ► CDF accumulation in bile canaliculi is inhibited by E2 or E17G in QTLI. ► Increasing exposure to E2 decreases CDF accumulation in bile canaliculi in QTLI. ► QTLI is feasible to assess Mrp2-based DDI involving drug metabolite in hepatocytes.

Natural occurrence of fungi and fungal metabolites in moldy tomatoes

DEFF Research Database (Denmark)

Andersen, B.; Frisvad, Jens Christian

2004-01-01

Fresh tomatoes, homegrown and from supermarkets, with developing fungal lesions were collected. Each lesion was sampled, and the resulting fungal cultures were identified morphologically, and extracted for analyzes of secondary metabolites. The tomatoes were incubated at 25 degreesC for a week....... extracted, and analyzed for fungal metabolites. Extracts from pure cultures were compared with extracts from the moldy tomatoes and fungal metabolite standards in two HPLC systems with DAD and FLD detection. The results showed that Penicillium tularense, Stemphylium eturmiunum. and S. cf. lycopersici were...

Revisiting the TCA cycle: signaling to tumor formation.

Science.gov (United States)

Raimundo, Nuno; Baysal, Bora E; Shadel, Gerald S

2011-11-01

A role for mitochondria in tumor formation is suggested by mutations in enzymes of the TCA cycle: isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH) and fumarate hydratase (FH). Although they are all components of the TCA cycle, the resulting clinical presentations do not overlap. Activation of the hypoxia pathway can explain SDH phenotypes, but recent data suggest that FH and IDH mutations lead to tumor formation by repressing cellular differentiation. In this review, we discuss recent findings in the context of both mitochondrial and cytoplasmic components of the TCA cycle, and we propose that extrametabolic roles of TCA cycle metabolites result in reduced cellular differentiation. Furthermore, activation of the pseudohypoxia pathway likely promotes the growth of these neoplasias into tumors. Copyright © 2011 Elsevier Ltd. All rights reserved.

Protein modification by acrolein: Formation and stability of cysteine adducts

OpenAIRE

Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

2009-01-01

The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

Biodegradation of clofibric acid and identification of its metabolites

Energy Technology Data Exchange (ETDEWEB)

Salgado, R. [REQUIMTE/CQFB, Chemistry Department, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); ESTS-IPS, Escola Superior de Tecnologia de Setubal do Instituto Politecnico de Setubal, Rua Vale de Chaves, Campus do IPS, Estefanilha, 2910-761 Setubal (Portugal); Oehmen, A. [REQUIMTE/CQFB, Chemistry Department, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Carvalho, G. [REQUIMTE/CQFB, Chemistry Department, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Instituto de Biologia Experimental e Tecnologica (IBET), Av. da Republica (EAN), 2784-505 Oeiras (Portugal); Noronha, J.P. [REQUIMTE/CQFB, Chemistry Department, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Reis, M.A.M., E-mail: amr@fct.unl.pt [REQUIMTE/CQFB, Chemistry Department, FCT, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)

2012-11-30

Graphical abstract: Metabolites produced during clofibric acid biodegradation. Highlights: Black-Right-Pointing-Pointer Clofibric acid is biodegradable. Black-Right-Pointing-Pointer Mainly heterotrophic bacteria degraded the clofibric acid. Black-Right-Pointing-Pointer Metabolites of clofibric acid biodegradation were identified. Black-Right-Pointing-Pointer The metabolic pathway of clofibric acid biodegradation is proposed. - Abstract: Clofibric acid (CLF) is the pharmaceutically active metabolite of lipid regulators clofibrate, etofibrate and etofyllinclofibrate, and it is considered both environmentally persistent and refractory. This work studied the biotransformation of CLF in aerobic sequencing batch reactors (SBRs) with mixed microbial cultures, monitoring the efficiency of biotransformation of CLF and the production of metabolites. The maximum removal achieved was 51% biodegradation (initial CLF concentration = 2 mg L{sup -1}), where adsorption and abiotic removal mechanisms were shown to be negligible, showing that CLF is indeed biodegradable. Tests showed that the observed CLF biodegradation was mainly carried out by heterotrophic bacteria. Three main metabolites were identified, including {alpha}-hydroxyisobutyric acid, lactic acid and 4-chlorophenol. The latter is known to exhibit higher toxicity than the parent compound, but it did not accumulate in the SBRs. {alpha}-Hydroxyisobutyric acid and lactic acid accumulated for a period, where nitrite accumulation may have been responsible for inhibiting their degradation. A metabolic pathway for the biodegradation of CLF is proposed in this study.

Generation of ER{alpha}-floxed and knockout mice using the Cre/LoxP system

Energy Technology Data Exchange (ETDEWEB)

Antonson, P., E-mail: per.antonson@ki.se [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Omoto, Y.; Humire, P. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Gustafsson, J.-A. [Department of Biosciences and Nutrition, Karolinska Institutet, Novum, SE-141 83 Huddinge (Sweden); Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States)

2012-08-10

Highlights: Black-Right-Pointing-Pointer ER{alpha} floxed and knockout mice were generated. Black-Right-Pointing-Pointer Disruption of the ER{alpha} gene results in sterility in both male and female mice. Black-Right-Pointing-Pointer ER{alpha}{sup -/-} mice have ovaries with hemorrhagic follicles and hypoplastic uterus. Black-Right-Pointing-Pointer Female ER{alpha}{sup -/-} mice develop obesity. -- Abstract: Estrogen receptor alpha (ER{alpha}) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ER{alpha} mouse line that can be used to knock out ER{alpha} in selected tissues by using the Cre/LoxP system. In this study, we established a new ER{alpha} knockout mouse line by crossing the floxed ER{alpha} mice with Cre deleter mice. Here we show that genetic disruption of the ER{alpha} gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ER{alpha} is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

Fate of cyanobacteria and their metabolites during water treatment sludge management processes

International Nuclear Information System (INIS)

Ho, Lionel; Dreyfus, Jennifer; Boyer, Justine; Lowe, Todd; Bustamante, Heriberto; Duker, Phil; Meli, Tass; Newcombe, Gayle

2012-01-01

Cyanobacteria and their metabolites are an issue for water authorities; however, little is known as to the fate of coagulated cyanobacterial-laden sludge during waste management processes in water treatment plants (WTPs). This paper provides information on the cell integrity of Anabaena circinalis and Cylindrospermopsis raciborskii during: laboratory-scale coagulation/sedimentation processes; direct filtration and backwashing procedures; and cyanobacterial-laden sludge management practices. In addition, the metabolites produced by A. circinalis (geosmin and saxitoxins) and C. raciborskii (cylindrospermopsin) were investigated with respect to their release (and possible degradation) during each of the studied processes. Where sedimentation was used, coagulation effectively removed cyanobacteria (and intracellular metabolites) without any considerable exertion on coagulant demand. During direct filtration experiments, cyanobacteria released intracellular metabolites through a stagnation period, suggesting that more frequent backwashing of filters may be required to prevent floc build-up and metabolite release. Cyanobacteria appeared to be protected within the flocs, with minimal damage during backwashing of the filters. Within coagulant sludge, cyanobacteria released intracellular metabolites into the supernatant after 3 d, even though cells remained viable up to 7 d. This work has improved the understanding of cyanobacterial metabolite risks associated with management of backwash water and sludge and is likely to facilitate improvements at WTPs, including increased monitoring and the application of treatment strategies and operational practices, with respect to cyanobacterial-laden sludge and/or supernatant recycle management. - Highlights: ► Coagulation removed cyanobacteria without an additional exertion on coagulant demand. ► During a stagnation period in direct filtration intracellular metabolites were released. ► Cyanobacterial cells were not damaged

Metabolite profiles and the risk of developing diabetes

OpenAIRE

2011-01-01

Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines, and other polar metabolites were profiled in baseline specimens using liquid chromatography-tandem mass spectrometry. Cases and controls were matched for age, body mass index and fasting g...

Synthesis of an Albendazole Metabolite: Characterization and HPLC Determination

Science.gov (United States)

Mahler, Graciela; Davyt, Danilo; Gordon, Sandra; Incerti, Marcelo; Nunez, Ivana; Pezaroglo, Horacio; Scarone, Laura; Serra, Gloria; Silvera, Mauricio; Manta, Eduardo

2008-01-01

In this laboratory activity, students are introduced to the synthesis of an albendazole metabolite obtained by a sulfide oxidation reaction. Albendazole as well as its metabolite, albendazole sulfoxide, are used as anthelmintic drugs. The oxidation reagent is H[subscript 2]O[subscript 2] in acetic acid. The reaction is environmental friendly,…

Tracer kinetic modelling of receptor data with mathematical metabolite correction

International Nuclear Information System (INIS)

Burger, C.; Buck, A.

1996-01-01

Quantitation of metabolic processes with dynamic positron emission tomography (PET) and tracer kinetic modelling relies on the time course of authentic ligand in plasma, i.e. the input curve. The determination of the latter often requires the measurement of labelled metabilites, a laborious procedure. In this study we examined the possibility of mathematical metabolite correction, which might obviate the need for actual metabolite measurements. Mathematical metabilite correction was implemented by estimating the input curve together with kinetic tissue parameters. The general feasibility of the approach was evaluated in a Monte Carlo simulation using a two tissue compartment model. The method was then applied to a series of five human carbon-11 iomazenil PET studies. The measured cerebral tissue time-activity curves were fitted with a single tissue compartment model. For mathematical metabolite correction the input curve following the peak was approximated by a sum of three decaying exponentials, the amplitudes and characteristic half-times of which were then estimated by the fitting routine. In the simulation study the parameters used to generate synthetic tissue time-activity curves (K 1 -k 4 ) were refitted with reasonable identifiability when using mathematical metabolite correciton. Absolute quantitation of distribution volumes was found to be possible provided that the metabolite and the kinetic models are adequate. If the kinetic model is oversimplified, the linearity of the correlation between true and estimated distribution volumes is still maintained, although the linear regression becomes dependent on the input curve. These simulation results were confirmed when applying mathematical metabolite correction to the 11 C iomazenil study. Estimates of the distribution volume calculated with a measured input curve were linearly related to the estimates calculated using mathematical metabolite correction with correlation coefficients >0.990. (orig./MG)

Quantitative quenching evaluation and direct intracellular metabolite analysis in Penicillium chrysogenum.

Science.gov (United States)

Meinert, Sabine; Rapp, Sina; Schmitz, Katja; Noack, Stephan; Kornfeld, Georg; Hardiman, Timo

2013-07-01

Sustained progress in metabolic engineering methodologies has stimulated new efforts toward optimizing fungal production strains such as through metabolite analysis of Penicillium chrysogenum industrial-scale processes. Accurate intracellular metabolite quantification requires sampling procedures that rapidly stop metabolism (quenching) and avoid metabolite loss via the cell membrane (leakage). When sampling protocols are validated, the quenching efficiency is generally not quantitatively assessed. For fungal metabolomics, quantitative biomass separation using centrifugation is a further challenge. In this study, P. chrysogenum intracellular metabolites were quantified directly from biomass extracts using automated sampling and fast filtration. A master/slave bioreactor concept was applied to provide industrial production conditions. Metabolic activity during sampling was monitored by 13C tracing. Enzyme activities were efficiently stopped and metabolite leakage was absent. This work provides a reliable method for P. chrysogenum metabolomics and will be an essential base for metabolic engineering of industrial processes. Copyright © 2013 Elsevier Inc. All rights reserved.

Extraction and applications of cyanotoxins and other cyanobacterial secondary metabolites.

Science.gov (United States)

Haque, Fatima; Banayan, Sara; Yee, Josephine; Chiang, Yi Wai

2017-09-01

The rapid proliferation of cyanobacteria in bodies of water has caused cyanobacterial blooms, which have become an increasing cause of concern, largely due to the presence of toxic secondary metabolites (or cyanotoxins). Cyanotoxins are the toxins produced by cyanobacteria that may be harmful to surrounding wildlife. They include hepatotoxins, neurotoxins and dermatotoxins, and are classified based on the organs they affect. There are also non-toxic secondary metabolites that include chelators and UV-absorbing compounds. This paper summarizes the optimal techniques for secondary metabolite extraction and the possible useful products that can be obtained from cyanobacteria, with additional focus given to products derived from secondary metabolites. It becomes evident that the potential for their use as biocides, chelators, biofuels, biofertilizers, pharmaceuticals, food and feed, and cosmetics has not yet been comprehensively studied or extensively implemented. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

Science.gov (United States)

Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

2018-02-01

Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites

Energy Technology Data Exchange (ETDEWEB)

Boll, M.; Weber, L.W.D.; Becker, E.; Stampfl, A. [Inst. of Toxicology, GSF - National Research Center for Environment and Health, Muenchen, Neuherberg (Germany)

2001-08-01

CCl{sub 4}-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl{sub 4} metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of {sup 14}C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl{sub 4}. (2) Low oxygen partial pressure increased the reductive metabolism of CCl{sub 4} and thus covalent binding. (3) [{sup 14}C]-CCl{sub 4} was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl{sub 4} revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe{sup 3+} and cumol hydroperoxide, respectively. (5) CCl{sub 4}, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E ({alpha}-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl{sub 4}-induced lipid peroxidation as well as covalent binding of CCl{sub 4} metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl{sub 3}{sup *} radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas the reaction with oxygen, to form CCl{sub 3}-OO{sup *}, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death. (orig.)

A Latex Metabolite Benefits Plant Fitness under Root Herbivore Attack

NARCIS (Netherlands)

Huber, M.; Epping, Janina; Schulze Gronover, C.; Fricke, Julia; Aziz, Zohra; Brillatz, Théo; Swyers, Michael; Kollner, T.G.; Vogel, H.; Hammerbacher, Almuth; Triebwasser-Freese, Daniella; Robert, Christelle A.M.; Verhoeven, K.J.F.; Preite, V.; Gershenzon, J.; Erb, M.

2016-01-01

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under

The reactive metabolite target protein database (TPDB)--a web-accessible resource.

Science.gov (United States)

Hanzlik, Robert P; Koen, Yakov M; Theertham, Bhargav; Dong, Yinghua; Fang, Jianwen

2007-03-16

The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins. The Reactive Metabolite Target Protein Database (TPDB) is a comprehensive, curated, searchable, documented compilation of publicly available information on the protein targets of reactive metabolites of 18 well-studied chemicals and drugs of known toxicity. TPDB software enables i) string searches for author names and proteins names/synonyms, ii) more complex searches by selecting chemical compound, animal species, target tissue and protein names/synonyms from pull-down menus, and iii) commonality searches over multiple chemicals. Tabulated search results provide information, references and links to other databases. The TPDB is a unique on-line compilation of information on the covalent modification of cellular proteins by reactive metabolites of chemicals and drugs. Its comprehensiveness and searchability should facilitate the elucidation of mechanisms of reactive metabolite toxicity. The database is freely available at http://tpdb.medchem.ku.edu/tpdb.html.

A validated HPLC-MS/MS assay for quantifying unstable pharmacologically active metabolites of clopidogrel in human plasma: application to a clinical pharmacokinetic study.

Science.gov (United States)

Furlong, Michael T; Savant, Ishani; Yuan, Moucun; Scott, Laura; Mylott, William; Mariannino, Thomas; Kadiyala, Pathanjali; Roongta, Vikram; Arnold, Mark E

2013-05-01

Clopidogrel is prescribed for the treatment of Acute Coronary Syndrome and recent myocardial infarction, recent stroke, or established peripheral arterial disease. A sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated to enable reliable quantification of four diastereomeric and chemically reactive thiol metabolites, two of which are pharmacologically active, in human plasma. The metabolites were stabilized by alkylation of their reactive thiol moieties with 2-bromo-3'-methoxyacetophenone (MPB). Following organic solvent mediated-protein precipitation in a 96-well plate format, chromatographic separation was achieved by gradient elution on an Ascentis Express RP-amide column. Chromatographic conditions were optimized to ensure separation of the four derivatized active metabolites. Derivatized metabolites and stable isotope-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The HPLC-MS/MS assay was validated over concentration ranges of 0.125-125 ng/mL for metabolites H1-H3 and 0.101-101 ng/mL for H4. Intra- and inter-assay precision values for replicate quality control samples were within 14.3% for all analytes during the assay validation. Mean quality control accuracy values were within ±6.3% of nominal values for all analytes. Assay recoveries were high (>79%). The four derivatized analytes were stable in human blood for at least 2 h at room temperature and on ice. The analytes were also stable in human plasma for at least 25 h at room temperature, 372 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of all four thiol metabolites in human plasma in support of a human pharmacokinetic study. Copyright © 2013 Elsevier B.V. All rights reserved.

Dietary Metabolites and Chronic Kidney Disease

Directory of Open Access Journals (Sweden)

Sho Hasegawa

2017-04-01

Full Text Available Dietary contents and their metabolites are closely related to chronic kidney disease (CKD progression. Advanced glycated end products (AGEs are a type of uremic toxin produced by glycation. AGE accumulation is not only the result of elevated glucose levels or reduced renal clearance capacity, but it also promotes CKD progression. Indoxyl sulfate, another uremic toxin derived from amino acid metabolism, accumulates as CKD progresses and induces tubulointerstitial fibrosis and glomerular sclerosis. Specific types of amino acids (d-serine or fatty acids (palmitate are reported to be closely associated with CKD progression. Promising therapeutic targets associated with nutrition include uremic toxin absorbents and inhibitors of AGEs or the receptor for AGEs (RAGE. Probiotics and prebiotics maintain gut flora balance and also prevent CKD progression by enhancing gut barriers and reducing uremic toxin formation. Nrf2 signaling not only ameliorates oxidative stress but also reduces elevated AGE levels. Bardoxolone methyl, an Nrf2 activator and NF-κB suppressor, has been tested as a therapeutic agent, but the phase 3 clinical trial was terminated owing to the high rate of cardiovascular events. However, a phase 2 trial has been initiated in Japan, and the preliminary analysis reveals promising results without an increase in cardiovascular events.

Formation of quinones by one-electron oxidation in the metabolism of benzo[a]pyrene and 6-fluorobenzo[a]pyrene

International Nuclear Information System (INIS)

Cavalieri, E.; Wong, A.; Cremonesi, P.; Warner, C.; Rogan, E.

1986-01-01

Metabolic activation of polycyclic aromatic hydrocarbons (PAH), as well as other chemical carcinogens, occurs by two major pathways: One-electron oxidation and two-electron oxidation, or monooxygenation. One-electron oxidation generates radical cations or radicals, depending on the molecule in which the oxidation occurs, whereas two-electron oxidation produces oxygenated metabolites. Radical cations of PAH are ultimate electrophilic metabolites capable of binding to cellular macromolecules to initiate the tumor process. In this paper the authors will provide evidence that one-electron oxidation is involved not only in PAH carcinogenesis, but also in the formation of certain metabolites. Metabolism of benzo[a]pyrene (BP) by cytochrome P-450 monooxygenase yields three classes of products: phenols, dihydrodiols and the quinones, 1,6-, 3,6- and 6,12- dione

Duodenal L cell density correlates with features of metabolic syndrome and plasma metabolites

Directory of Open Access Journals (Sweden)

Annieke C G van Baar

2018-05-01

Full Text Available Background: Enteroendocrine cells are essential for the regulation of glucose metabolism, but it is unknown whether they are associated with clinical features of metabolic syndrome (MetS and fasting plasma metabolites. Objective: We aimed to identify fasting plasma metabolites that associate with duodenal L cell, K cell and delta cell densities in subjects with MetS with ranging levels of insulin resistance. Research design and methods: In this cross-sectional study, we evaluated L, K and delta cell density in duodenal biopsies from treatment-naïve males with MetS using machine-learning methodology. Results: We identified specific clinical biomarkers and plasma metabolites associated with L cell and delta cell density. L cell density was associated with increased plasma metabolite levels including symmetrical dimethylarginine, 3-aminoisobutyric acid, kynurenine and glycine. In turn, these L cell-linked fasting plasma metabolites correlated with clinical features of MetS. Conclusions: Our results indicate a link between duodenal L cells, plasma metabolites and clinical characteristics of MetS. We conclude that duodenal L cells associate with plasma metabolites that have been implicated in human glucose metabolism homeostasis. Disentangling the causal relation between L cells and these metabolites might help to improve the (small intestinal-driven pathophysiology behind insulin resistance in human obesity.

Significance of metabolites in the environmental risk assessment of pharmaceuticals consumed by human.

Science.gov (United States)

Han, Eun Jeong; Lee, Dong Soo

2017-08-15