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  • 1.
    Chen, Yang
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Oliw, Ernst
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism2017In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 625-626, p. 24-29Article in journal (Refereed)
    Abstract [en]

    Plants and fungi form jasmonic acid from a-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-Iipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8 and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of alpha-linolenic and linoleic acids to allene oxides/alpha-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues.

  • 2. Elshorbagy, A K
    et al.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Samocha-Bonet, D
    Refsum, H
    Heilbronn, L K
    Serum S-adenosylmethionine, but not methionine, increases in response to overfeeding in humans.2016In: Nutrition & Diabetes, ISSN 2044-4052, E-ISSN 2044-4052, Vol. 6, article id e192Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Plasma concentration of the methyl donor S-adenosylmethionine (SAM) is linearly associated with body mass index (BMI) and fat mass. As SAM is a high-energy compound and a sensor of cellular nutrient status, we hypothesized that SAM would increase with overfeeding.

    METHODS: Forty normal to overweight men and women were overfed by 1250 kcal per day for 28 days.

    RESULTS: Serum SAM increased from 106 to 130 nmol/l (P=0.006). In stratified analysis, only those with weight gain above the median (high-weight gainers; average weight gain 3.9±0.3 kg) had increased SAM (+42%, P=0.001), whereas low-weight gainers (weight gain 1.5±0.2 kg) did not (Pinteraction=0.018). Overfeeding did not alter serum concentrations of the SAM precursor, methionine or the products, S-adenosyl-homocysteine and homocysteine. The SAM/SAH (S-adenosylhomocysteine) ratio was unchanged in the total population, but increased in high-weight gainers (+52%, P=0.006, Pinteraction =0.005). Change in SAM correlated positively with change in weight (r=0.33, P=0.041) and fat mass (r=0.44, P=0.009), but not with change in protein intake or plasma methionine, glucose, insulin or low-density lipoprotein (LDL)-cholesterol.

    CONCLUSION: Overfeeding raised serum SAM in proportion to the fat mass gained. The increase in SAM may help stabilize methionine levels, and denotes a responsiveness of SAM to nutrient state in humans. The role of SAM in human energy metabolism deserves further attention.

  • 3. Elshorbagy, Amany
    et al.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Basta, Marianne
    Basta, Caroline
    Turner, Cheryl
    Khaled, Maram
    Refsum, Helga
    Amino acid changes during transition to a vegan diet supplemented with fish in healthy humans.2017In: European Journal of Nutrition, ISSN 1436-6207, E-ISSN 1436-6215, Vol. 56, no 5, p. 1953-1962Article in journal (Refereed)
    Abstract [en]

    PURPOSE: To explore whether changes in dietary protein sources can lower plasma branched-chain amino acids (BCAAs), aromatic amino acids and sulfur amino acids (SAAs) that are often elevated in the obese, insulin-resistant state and in type 2 diabetes.

    METHODS: Thirty-six subjects (mean age 31 ± 2 years) underwent a voluntary abstinence from meat, poultry, eggs, and dairy products for 6 weeks, while enriching the diet with fish, in fulfillment of a religious fast. Subjects were assessed 1 week before the fast (V1), 1 week after initiation of the fast (V2) and in the last week of the fast (V3). Thirty-four subjects completed all three visits.

    RESULTS: Fasting plasma BCAAs decreased at V2 and remained low at V3 (P < 0.001 for all). Valine showed the greatest decline, by 20 and 19 % at V2 and V3, respectively. Phenylalanine and tryptophan, but not tyrosine, also decreased at V2 and V3. The two proteinogenic SAAs, methionine and cysteine, remained stable, but the cysteine product, taurine, decreased from 92 ± 7 μmol/L to 66 ± 6 (V2; P = 0.003) and 65 ± 6 μmol/L (V3; P = 0.003). A progressive decline in plasma glutamic acid, coupled with an increase in glutamine, was observed. Plasma total and LDL cholesterol decreased at V2 and V3 (P < 0.001 for all).

    CONCLUSION: Changing dietary protein sources to plant- and fish-based sources in an ad libitum setting lowers the plasma BCAAs that have been linked to diabetes risk. These findings point to habitual diet as a potentially modifiable determinant of fasting plasma BCAA concentrations.

  • 4. Elshorbagy, Amany K
    et al.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Scudamore, Cheryl L
    McMurray, Fiona
    Cater, Heather
    Hough, Tertius
    Cox, Roger
    Refsum, Helga
    Exploring the Lean Phenotype of Glutathione-Depleted Mice: Thiol, Amino Acid and Fatty Acid Profiles.2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0163214Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models.

    OBJECTIVE: To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice.

    DESIGN: Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis).

    RESULTS: Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all).

    CONCLUSION: Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism.

  • 5.
    Farina, Nicolas
    et al.
    Brighton & Sussex Med Sch, Ctr Dementia Studies, Brighton BN1 9RY, E Sussex, England..
    Jernerén, Fredrik
    Univ Oxford, Dept Pharmacol, Oxford OX1 3QT, England.;Uppsala Univ, Dept Pharmaceut Biosci, S-75237 Uppsala, Sweden..
    Turner, Cheryl
    Univ Oxford, Dept Pharmacol, Oxford OX1 3QT, England..
    Hart, Kathryn
    Univ Surrey, Dept Nutrit Sci, Guildford GU2 7XH, Surrey, England..
    Tabet, Naji
    Brighton & Sussex Med Sch, Ctr Dementia Studies, Brighton BN1 9RY, E Sussex, England.;Sussex Partnership NHS Fdn Trust, Dementia Res Unit, Crowborough TN6 1HB, England..
    Homocysteine concentrations in the cognitive progression of Alzheimer's disease2017In: Experimental Gerontology, ISSN 0531-5565, E-ISSN 1873-6815, Vol. 99, p. 146-150Article in journal (Refereed)
    Abstract [en]

    Objectives: Hyperhomocysteinemia in Alzheimer's disease (AD) is widely reported and appears to worsen as the disease progresses. While active dietary intervention with vitamins B12 and folate decreases homocysteine blood levels, with promising clinical outcomes in Mild Cognitive Impairment (MCI), this so far has not been replicated in established AD populations. The aim of the study is to explore the relationship between hyperhomocystenemia and relevant vitamins as the disease progresses. Methods: In this longitudinal cohort study, 38 participants with mild to moderate AD were followed for an average period of 13 months. Plasma folate, vitamin B12 and homocysteine concentrations were measured at baseline and at follow-up. Dietary intake of B vitamins was also measured. Spearman's correlations were conducted by homocysteine and B vitamin status. Results: As expected, cognitive status significantly declined over the follow-up period and this was paralleled by a significant increase in homocysteine concentrations (p = 0.006). However, during this follow-up period there was no significant decline in neither dietary intake, nor the corresponding blood concentrations of vitamin B12/folate, with both remaining within normal values. Changes in blood concentrations of B vitamins were not associated with changes in homocysteine levels (p > 0.05). Conclusion: In this study, the increase in homocysteine observed in AD patients as the disease progresses cannot be solely explained by dietary and blood levels of folate and vitamin B12. Other dietary and non-dietary factors may contribute to hyperhomocysteinemia and its toxic effect in AD, which needs to be explored to optimise timely intervention strategies.

  • 6.
    Garscha, Ulrike
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Chung, D.
    Keller, N.P.
    Hamberg, M.
    Oliw, Ernst H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 48, p. 34707-34718Article in journal (Refereed)
    Abstract [en]

    Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z- hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

  • 7. Haj-Yasein, Nadia Nabil
    et al.
    Berg, Ole
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Refsum, Helga
    Nebb, Hilde I
    Dalen, Knut Tomas
    Cysteine deprivation prevents induction of peroxisome proliferator-activated receptor gamma-2 and adipose differentiation of 3T3-L1 cells.2017In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1862, no 6, p. 623-635, article id S1388-1981(17)30033-1Article in journal (Refereed)
    Abstract [en]

    Plasma cysteine is strongly associated with body fat mass in human cohorts and diets low in cysteine prevents fat accumulation in mice. It is unclear if plasma cysteine affects fat development or if fat accumulation raises plasma cysteine. To determine if cysteine affects adipogenesis, we differentiated 3T3-L1 preadipocytes in medium with reduced cysteine. Cells incubated in media with 10-20μM cysteine exhibited reduced capacity to differentiate into triacylglycerol-storing mature adipocytes compared with cells incubated with 50μM cysteine. Low cysteine severely reduced expression of peroxisome proliferator-activated receptor gamma2 (Pparγ2) and its target genes perlipin1 (Plin1) and fatty acid binding protein-4 (Fabp4). Expression of stearoyl-CoA desaturase-1 (Scd1), known to be repressed with cysteine depletion, was also reduced with low cysteine. Medium depletion of the essential amino acids leucine, valine, and isoleucine had only a modest effect on adipocyte specific gene expression and differentiation. Stimulation with the PPARγ agonist BRL-49653 or addition of a hydrogen sulfide donor enhanced differentiation of 3T3-L1 cells cultured in low cysteine. This demonstrates that the ability to induce PPARγ expression is preserved when cells are cultured in low cysteine. It therefore appears that cysteine depletion inhibits adipogenesis by specifically affecting molecular pathways required for induction of PPARγ expression, rather than through a general reduction of global protein synthesis. In conclusion, we show that low extracellular cysteine reduces adipocyte differentiation by interfering with PPARγ2 and PPARγ target gene expression. Our results provide further evidence for the hypothesis that plasma cysteine is a casual determinant for body fat mass.

  • 8.
    Jerneren, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Oliw, Ernst H.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    The Fatty Acid 8,11-Diol Synthase of Aspergillus fumigatus is Inhibited by Imidazole Derivatives and Unrelated to PpoB2012In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 47, no 7, p. 707-717Article in journal (Refereed)
    Abstract [en]

    (8R)-Hydroperoxy-(9Z,12Z)-octadecadienoic acid (8-HPODE) is formed by aspergilli as an intermediate in biosynthesis of oxylipins with effects on sporulation. 8-HPODE is transformed by separate diol synthases to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxy-(9Z,12Z)-octadecadienoic acids (5,8- and 8,11-DiHODE). The former is formed by the cytochrome P450 (P450) domain of 5,8-linoleate diol synthase (5,8-LDS or PpoA). Our aim was to characterize the 8,11-diol synthase of Aspergillus fumigatus, which is prominent in many strains. The 8,11-diol synthase was soluble and had a larger molecular size (>100 kDa) than most P450. Miconazole, ketoconazole, and 1-benzylimidazole, classical inhibitors of P450, reduced the biosynthesis of 8,11-DiHODE from 8-HPODE (apparent IC50 values similar to 0.8, similar to 5, and similar to 0.6 mu M, respectively), but did not inhibit the biosynthesis of 5,8-DiHODE. Analysis of hydroperoxides of regioisomeric C-18 and C-20 fatty acids showed that the 8,11-diol synthase was specific for certain hydroperoxides with R configuration. The suprafacial hydrogen abstraction and oxygen insertion at C-11 of 8-HPODE was associated with a small deuterium kinetic isotope effect ((H)k(cat)/(D)k(cat) similar to 1.5), consistent with P450-catalyzed oxidation. The genome of A. fumigatus contains over 70 P450 sequences. The reaction mechanism, size, and solubility of 8,11-diol synthase pointed to PpoB, a homologue of 5,8-LDS, as a possible candidate of this activity. Gene deletion of ppoB of A. fumigatus strains AF:Delta ku80 and J272 did not inhibit biosynthesis of 8,11-DiHODE and recombinant PpoB appeared to lack diol synthase activity. We conclude that 8,11-DiHODE is formed from 8-HPODE by a soluble and substrate-specific 8,11-diol synthase with catalytic characteristics of class III P450.

  • 9.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Novel Fatty Acid Dioxygenases of Human and Plant Pathogenic Fungi: Studies by Gene Deletion and Expression2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The dioxygenase-cytochrome P450 fusion proteins (DOX-CYP) comprise a heme-containing enzyme family that shares structural and catalytic properties with mammalian prostaglandin H (PGH) synthases. 7,8-Linoleate diol synthase (7,8-LDS) of Gaeumannomyces graminis was first characterized, and DOX-CYP enzymes are of mechanistic and biological interest. The growing number of fungal genome sequences has revealed DOX-CYP homologues in medically and economically important species. The aim of this thesis was to identify novel members of the DOX-CYP fusion protein family.

    The devastating rice pathogen Magnaporthe oryzae contains two DOX-CYP genes. The fungus synthesizes 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by dioxygenation of linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8R-HPODE), and subsequent isomerisation to the diol. 7,8-LDS of M. oryzae was identified by gene deletion, but the infection and reproduction processes of the Δ7,8-LDS strain were not altered. A mutant with constitutive protein kinase A activity profoundly changed the oxygenation profile, possibly due to post-translational modification.

    The human pathogens Aspergillus fumigatus and A. clavatus contain three DOX-CYP, designated psi producing oxygenase A (ppoA), ppoB, and ppoC, and form three oxylipins: 5S,8R-DiHODE, 8R,11S-DiHODE, and 10R-hydroxyoctadecadienoic acid.  PpoA was identified as 5,8-LDS, and ppoC as 10R-DOX. The 8,11-linoleate hydroperoxide isomerase activity was reduced by two imidazole-containing P450 inhibitors, miconazole and 1-benzylimidazole. PpoB could not be linked to the biosynthesis of 8,11-DiHODE for the following reasons: First, the 8,11-hydroperoxide isomerase activity was retained in A. fumigatus ΔppoB strains. Second, the P450 domain of the deduced ppoB of A. clavatus lacks a heme-thiolate cysteine ligand, presumably essential for hydroperoxide isomerase activity.

    Linoleate 9R-DOX activities of Aspergillus terreus and Lasiodiplodia theobromae were discovered. 9R-HPODE was further converted into unstable allene oxides, as judged by the accumulation of their hydrolysis products, α- and γ-ketols. These allene oxide synthase activities were specific for 9R-hydroperoxides. The 9R-DOX and AOS were found to have unique characteristics.

    In conclusion, novel DOX-CYP enzymes were identified in human and plant pathogenic fungi. These enzymes might be involved in biological processes, and show interesting catalytic similarities to human PGH synthase and thromboxane synthase (CYP5A).

    List of papers
    1. Gene deletion of 7,8-linoleate diol synthase of the rice blast fungus: studies on pathogenicity, stereochemistry, and oxygenation mechanisms
    Open this publication in new window or tab >>Gene deletion of 7,8-linoleate diol synthase of the rice blast fungus: studies on pathogenicity, stereochemistry, and oxygenation mechanisms
    Show others...
    2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 8, p. 5308-5316Article in journal (Refereed) Published
    Abstract [en]

    Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Delta7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Delta7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Delta mac1 sum1-99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.

    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-122730 (URN)10.1074/jbc.M109.062810 (DOI)000275327200024 ()20023302 (PubMedID)
    Available from: 2010-04-20 Created: 2010-04-16 Last updated: 2018-01-12Bibliographically approved
    2. Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism
    Open this publication in new window or tab >>Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism
    Show others...
    2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 48, p. 34707-34718Article in journal (Refereed) Published
    Abstract [en]

    Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z- hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-15043 (URN)10.1074/jbc.M705366200 (DOI)000251155200013 ()17906293 (PubMedID)
    Available from: 2008-02-04 Created: 2008-02-04 Last updated: 2018-01-12Bibliographically approved
    3. Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain: a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
    Open this publication in new window or tab >>Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain: a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase
    2011 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 506, no 2, p. 216-222Article in journal (Refereed) Published
    Abstract [en]

    5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10R-dioxygenase. We conclude that ∼675 amino acids are sufficient to support 8-DOX activity.

    Keywords
    Cytochrome P450, fusion protein, heme-dependent peroxidase, hydroperoxide isomerase, LC-MS/MS, oxylipins
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Research subject
    Biochemical Pharmacology
    Identifiers
    urn:nbn:se:uu:diva-142985 (URN)10.1016/j.abb.2010.11.022 (DOI)000286961600014 ()21130068 (PubMedID)
    Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2017-12-11Bibliographically approved
    4. Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus
    Open this publication in new window or tab >>Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus
    Show others...
    2010 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1801, no 4, p. 503-507Article in journal (Refereed) Published
    Abstract [en]

    Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity ( approximately 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain ( approximately 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of Aspergillusnidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with >65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.

    National Category
    Medical and Health Sciences Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-122729 (URN)10.1016/j.bbalip.2009.12.012 (DOI)000275590100013 ()20045744 (PubMedID)
    Available from: 2010-04-20 Created: 2010-04-16 Last updated: 2018-01-12Bibliographically approved
    5. The 8,11-linoleate hydroperoxide isomerase activity of Aspergillus fumigatus is unaffected by gene deletion of ppoB  and inhibited by miconazole and benzylimidazole.
    Open this publication in new window or tab >>The 8,11-linoleate hydroperoxide isomerase activity of Aspergillus fumigatus is unaffected by gene deletion of ppoB  and inhibited by miconazole and benzylimidazole.
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-142941 (URN)
    Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2011-03-11
    6. Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus
    Open this publication in new window or tab >>Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus
    2010 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 495, no 1, p. 67-73Article in journal (Refereed) Published
    Abstract [en]

    Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-2H]18:2n-6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (alpha-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (gamma-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. alpha-Linolenic acid and 20:2n-6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but alpha- and gamma-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.

    Keywords
    Catalase, Cytochrome P450, 9R-HpODE, Heme peroxidase, Jasmonic acid, Oxygenation mechanism, Potato stolons
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-122731 (URN)10.1016/j.abb.2009.12.022 (DOI)000275137000011 ()20043865 (PubMedID)
    Available from: 2010-04-20 Created: 2010-04-16 Last updated: 2018-01-12Bibliographically approved
    7. Fatty acid dioxygenase and allene oxide synthase activities of Lasiodiplodia theobromae. A comparison with Aspergillus terreus.
    Open this publication in new window or tab >>Fatty acid dioxygenase and allene oxide synthase activities of Lasiodiplodia theobromae. A comparison with Aspergillus terreus.
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-142942 (URN)
    Available from: 2011-01-18 Created: 2011-01-18 Last updated: 2011-03-11
  • 10.
    Jernerén, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Elshorbagy, Amany K
    Oulhaj, Abderrahim
    Smith, Stephen M
    Refsum, Helga
    Smith, A David
    Brain atrophy in cognitively impaired elderly: the importance of long-chain ω-3 fatty acids and B vitamin status in a randomized controlled trial.2015In: American Journal of Clinical Nutrition, ISSN 0002-9165, E-ISSN 1938-3207, Vol. 102, no 1, p. 215-21Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Increased brain atrophy rates are common in older people with cognitive impairment, particularly in those who eventually convert to Alzheimer disease. Plasma concentrations of omega-3 (ω-3) fatty acids and homocysteine are associated with the development of brain atrophy and dementia.

    OBJECTIVE: We investigated whether plasma ω-3 fatty acid concentrations (eicosapentaenoic acid and docosahexaenoic acid) modify the treatment effect of homocysteine-lowering B vitamins on brain atrophy rates in a placebo-controlled trial (VITACOG).

    DESIGN: This retrospective analysis included 168 elderly people (≥70 y) with mild cognitive impairment, randomly assigned either to placebo (n = 83) or to daily high-dose B vitamin supplementation (folic acid, 0.8 mg; vitamin B-6, 20 mg; vitamin B-12, 0.5 mg) (n = 85). The subjects underwent cranial magnetic resonance imaging scans at baseline and 2 y later. The effect of the intervention was analyzed according to tertiles of baseline ω-3 fatty acid concentrations.

    RESULTS: There was a significant interaction (P = 0.024) between B vitamin treatment and plasma combined ω-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) on brain atrophy rates. In subjects with high baseline ω-3 fatty acids (>590 μmol/L), B vitamin treatment slowed the mean atrophy rate by 40.0% compared with placebo (P = 0.023). B vitamin treatment had no significant effect on the rate of atrophy among subjects with low baseline ω-3 fatty acids (<390 μmol/L). High baseline ω-3 fatty acids were associated with a slower rate of brain atrophy in the B vitamin group but not in the placebo group.

    CONCLUSIONS: The beneficial effect of B vitamin treatment on brain atrophy was observed only in subjects with high plasma ω-3 fatty acids. It is also suggested that the beneficial effect of ω-3 fatty acids on brain atrophy may be confined to subjects with good B vitamin status. The results highlight the importance of identifying subgroups likely to benefit in clinical trials. This trial was registered at www.controlled-trials.com as ISRCTN94410159.

  • 11.
    Jernerén, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Eng, Felipe
    Hamberg, Mats
    Oliw, Ernst H
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Linolenate 9R-Dioxygenase and Allene Oxide Synthase Activities of Lasiodiplodia theobromae2012In: Lipids, ISSN 0024-4201, E-ISSN 1558-9307, Vol. 47, no 1, p. 65-73Article in journal (Refereed)
    Abstract [en]

    Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2 mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-(2)H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200 mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.

  • 12.
    Jernerén, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hamberg, Mats
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet.
    Oliw, Ernst
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fatty acid dioxygenase and allene oxide synthase activities of Lasiodiplodia theobromae. A comparison with Aspergillus terreus.Manuscript (preprint) (Other academic)
  • 13.
    Jernerén, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Oliw, Enst
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    The 8,11-linoleate hydroperoxide isomerase activity of Aspergillus fumigatus is unaffected by gene deletion of ppoB  and inhibited by miconazole and benzylimidazole.Manuscript (preprint) (Other academic)
  • 14.
    Oliw, Ernst H.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hoffmann, Inga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Sahlin, Margareta
    Molekylärbiologi, Stockholms universitet.
    Garscha, Ulrike
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms2011In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1811, no 3, p. 138-147Article in journal (Refereed)
    Abstract [en]

    Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of ~88 and ~87kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III)OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.

  • 15.
    Oliw, Ernst H.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wennman, Anneli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hoffmann, Inga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Garscha, Ulrike
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hamberg, Mats
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases2011In: Journal of Lipid Research, ISSN 0022-2275, E-ISSN 1539-7262, Vol. 52, no 11, p. 1995-2004Article in journal (Refereed)
    Abstract [en]

    Seven Z-octadecenoic acids having the double bond located in positions 6Z to 13Z were photooxidized. The resulting hydroperoxy-E-octadecenoic acids [HpOME(E)] were resolved by chiral phase-HPLC-MS, and the absolute configurations of the enantiomers were determined by gas chromatographic analysis of diastereoisomeric derivatives. The MS/MS/MS spectra showed characteristic fragments, which were influenced by the distance between the hydroperoxide and carboxyl groups. These fatty acids were then investigated as substrates of cyclooxygenase-1 (COX-1), manganese lipoxygenase (MnLOX), and the (8R)-dioxygenase (8R-DOX) activities of two linoleate diol synthases (LDS) and 10R-DOX. COX-1 and MnLOX abstracted hydrogen at C-11 of (12Z)-18:1 and C-12 of (13Z)-18:1. (11Z)-18:1 was subject to hydrogen abstraction at C-10 by MnLOX and at both allylic positions by COX-1. Both allylic hydrogens of (8Z)-18:1 were also abstracted by 8R-DOX activities of LDS and 10R-DOX, but only the allylic hydrogens close to the carboxyl groups of (11Z)-18:1 and (12Z)-18:1. 8R-DOX also oxidized monoenoic C(14)-C(20) fatty acids with double bonds at the (9Z) position, suggesting that the length of the omega end has little influence on positioning for oxygenation. We conclude that COX-1 and MnLOX can readily abstract allylic hydrogens of octadecenoic fatty acids from C-10 to C-12 and 8R-DOX from C-7 and C-12.

  • 16. Oulhaj, Abderrahim
    et al.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Refsum, Helga
    Smith, A David
    de Jager, Celeste A
    Omega-3 Fatty Acid Status Enhances the Prevention of Cognitive Decline by B Vitamins in Mild Cognitive Impairment.2016In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 50, no 2, p. 547-57Article in journal (Refereed)
    Abstract [en]

    A randomized trial (VITACOG) in people with mild cognitive impairment (MCI) found that B vitamin treatment to lower homocysteine slowed the rate of cognitive and clinical decline. We have used data from this trial to see whether baseline omega-3 fatty acid status interacts with the effects of B vitamin treatment. 266 participants with MCI aged ≥70 years were randomized to B vitamins (folic acid, vitamins B6 and B12) or placebo for 2 years. Baseline cognitive test performance, clinical dementia rating (CDR) scale, and plasma concentrations of total homocysteine, total docosahexaenoic and eicosapentaenoic acids (omega-3 fatty acids) were measured. Final scores for verbal delayed recall, global cognition, and CDR sum-of-boxes were better in the B vitamin-treated group according to increasing baseline concentrations of omega-3 fatty acids, whereas scores in the placebo group were similar across these concentrations. Among those with good omega-3 status, 33% of those on B vitamin treatment had global CDR scores >0 compared with 59% among those on placebo. For all three outcome measures, higher concentrations of docosahexaenoic acid alone significantly enhanced the cognitive effects of B vitamins, while eicosapentaenoic acid appeared less effective. When omega-3 fatty acid concentrations are low, B vitamin treatment has no effect on cognitive decline in MCI, but when omega-3 levels are in the upper normal range, B vitamins interact to slow cognitive decline. A clinical trial of B vitamins combined with omega-3 fatty acids is needed to see whether it is possible to slow the conversion from MCI to AD.

  • 17.
    Pierozan, Paula
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ransome, Yusuf
    Karlsson, Oskar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.
    The Choice of Euthanasia Method Affects Metabolic Serum Biomarkers2017In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 21, p. 113-118Article in journal (Refereed)
    Abstract [en]

    The impact of euthanasia methods on endocrine and metabolic parameters in rodent tissues and biological fluids is highly relevant for the accuracy and reliability of the data collected. However, few studies concerning this issue are found in the literature. We compared the effects of three euthanasia methods currently used in animal experimentation (i.e. decapitation, CO2 inhalation and pentobarbital injection) on the serum levels of corticosterone, insulin, glucose, triglycerides, cholesterol and a range of free fatty acids in rats. The corticosterone and insulin levels were not significantly affected by the euthanasia protocol used. However, euthanasia by an overdose of pentobarbital (120 mg/kg intraperitoneal injection) increased the serum levels of glucose, and decreased cholesterol, stearic and arachidonic acids levels compared with euthanasia by CO2 inhalation and decapitation. CO2 inhalation appears to increase the serum levels of triglycerides, while euthanasia by decapitation induced no individual discrepant biomarker level. We conclude that choice of the euthanasia methods is critical for the reliability of serum biomarkers and indicate the importance of selecting adequate euthanasia methods for metabolic analysis in rodents. Decapitation without anaesthesia may be the most adequate method of euthanasia when taking both animal welfare and data quality in consideration.

  • 18.
    Wennman, Anneli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hamberg, Mats
    Karolinska institutet.
    Oliw, Ernst H
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 38, p. 31757-31765Article in journal (Refereed)
    Abstract [en]

    Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (∼80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ∼100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.

  • 19. Zhang, Weihua
    et al.
    Jernerén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Dept. of Pharmacology, University of Oxford.
    Lehne, Benjamin C
    Chen, Ming-Huei
    Luben, Robert N
    Johnston, Carole
    Elshorbagy, Amany
    Eppinga, Ruben N
    Scott, William R
    Adeyeye, Elizabeth
    Scott, James
    Böger, Rainer H
    Khaw, Kay-Tee
    van der Harst, Pim
    Wareham, Nicholas J
    Vasan, Ramachandran S
    Chambers, John C
    Refsum, Helga
    Kooner, Jaspal S
    Genome-wide association reveals that common genetic variation in the kallikrein-kinin system is associated with serum L-arginine levels.2016In: Thrombosis and haemostasis, ISSN 2567-689X, Vol. 116, no 6, p. 1041-1049Article in journal (Refereed)
    Abstract [en]

    ). Together these two loci explain 7 % of the total variance in serum L-arginine concentrations. The associations at both loci were replicated in independent cohorts with plasma L-arginine measurements (p<0.004). The two sentinel SNPs are in nearly complete LD with the nonsynonymous SNP rs3733402 at KLKB1 and the 5'-UTR SNP rs1801020 at F12, respectively. SNPs at both loci are associated with blood pressure. Our findings provide new insight into the genetic regulation of L-arginine and its potential relationship with cardiovascular risk.

1 - 19 of 19
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