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  • 1.
    Ahlgren, Joakim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Tranvik, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Evolution, Limnology.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Waldebäck, Monica
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Markides, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
    Rydin, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Evolution, Limnology.
    Sediment Depth Attenuation of Biogenic Phosphorus Compounds Measured by 31P NMR2005In: Environmental Science and Technology, ISSN 0013-936X, E-ISSN 1520-5851, Vol. 39, no 3, p. 867-872Article in journal (Refereed)
    Abstract [en]

    Being a major cause of eutrophication and subsequent loss of water quality, the turnover of phosphorus (P) in lake sediments is in need of deeper understanding. A major part of the flux of P to eutrophic lake sediments is organically bound or of biogenic origin. This P is incorporated in a poorly described mixture of autochthonous and allochthonous sediment and forms the primary storage of P available for recycling to the water column, thus regulating lake trophic status. To identify and quantify biogenic sediment P and assess its lability, we analyzed sediment cores from Lake Erken, Sweden, using traditional P fractionation, and in parallel, NaOH extracts were analyzed using 31P NMR. The surface sediments contain orthophosphates (ortho-P) and pyrophosphates (pyro-P), as well as phosphate mono- and diesters. The first group of compounds to disappear with increased sediment depth is pyrophosphate, followed by a steady decline of the different ester compounds. Estimated half-life times of these compound groups are about 10 yr for pyrophosphate and 2 decades for mono- and diesters. Probably, these compounds will be mineralized to ortho-P and is thus potentially available for recycling to the water column, supporting further growth of phytoplankton. In conclusion, 31P NMR is a useful tool to asses the bioavailability of certain P compound groups, and the combination with traditional fractionation techniques makes quantification possible.

  • 2.
    Andersson, David C.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Martinez, N.
    Zeller, D.
    Rondahl, S. H.
    Koza, M. M.
    Frick, B.
    Ekstrom, F.
    Peters, J.
    Linusson, Anna
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Changes in dynamics of alpha-chymotrypsin due to covalent inhibitors investigated by elastic incoherent neutron scattering2017In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 19, no 37, p. 25369-25379Article in journal (Refereed)
    Abstract [en]

    An essential role of enzymes is to catalyze various chemical reactions in the human body and inhibition of the enzymatic activity by small molecules is the mechanism of action of many drugs or tool compounds used to study biological processes. Here, we investigate the effect on the dynamics of the serine protease alpha-chymotrypsin when in complex with two different covalently bound inhibitors using elastic incoherent neutron scattering. The results show that the inhibited enzyme displays enhanced dynamics compared to the free form. The difference was prominent at higher temperatures (240-310 K) and the type of motions that differ include both small amplitude motions, such as hydrogen atom rotations around a methyl group, and large amplitude motions, such as amino acid side chain movements. The measurements were analyzed with multivariate methods in addition to the standard univariate methods, allowing for a more in-depth analysis of the types of motions that differ between the two forms. The binding strength of an inhibitor is linked to the changes in dynamics occurring during the inhibitor-enzyme binding event and thus these results may aid in the deconvolution of this fundamental event and in the design of new inhibitors.

  • 3. Ashitani, T.
    et al.
    Kusumoto, N.
    Borg-Karlson, Anna Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Fujita, K.
    Takahashi, K.
    Antitermite activity of β-caryophyllene epoxide and episulfide2013In: Zeitschrift für Naturforschung C - A Journal of Biosciences, ISSN 0939-5075, E-ISSN 1865-7125, Vol. 68 C, no 7-8, p. 302-306Article in journal (Refereed)
    Abstract [en]

    Caryophyllene-6,7-epoxide and caryophyllene-6,7-episulfide can be easily synthesized from β-caryophyllene by autoxidation or episulfidation. The bioactivities of β-caryophyllene and its derivatives were investigated against the subterranean termite Reticulitermes speratus Kolbe. The antifeedant, feeding, and termiticidal activities of each compound were tested using no-choice, dual-choice, and non-contact methods. Antitermitic activities were not shown by β-caryophyllene, but were observed for the oxide and sulfide derivatives. Caryophyllene- 6,7-episulfide showed especially high antifeedant and termiticidal activities. Thus, naturally abundant, non-bioactive β-caryophyllene can be easily converted into an antitermite reagent via a non-biological process.

  • 4.
    Axelsson, Karolin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Chemical signals in interactions between Hylobius abietis and associated bacteria2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The pine weevil (Hylobius abietis L.) is one of the two topmost economically important insect pests in Swedish conifer forests. The damage increase in areas were the silvicultural practice is to use clear cuttings were the insects gather and breed. During egglaying the female protects her offspring by creating a cave in roots and stumps were she puts her egg and covers it with frass, a mixture of weevil feces and chewed bark. Adult pine weevils have been observed to feed on the other side of the egg laying site and antifeedant substance has been discovered in the feces of the pine weevil. We think it is possible that microorganisms present in the frass contribute with antifeedant/repellent substances. Little is known about the pine weevils associated bacteria community and their symbiotic functions. In this thesis the bacterial community is characterized in gut and frass both from pine weevils in different populations across Europe as well as after a 28 day long diet regime on Scots pine, silver birch or bilberry. Volatile substances produced by isolated bacteria as well as from a consortium of microorganisms were collected with solid phase micro extraction (SPME) and analyzed with GC-MS. The main volatiles were tested against pine weevils using a two-choice test. Wolbachia, Rahnella aquatilis, Serratia and Pseudomonas syringae was commonly associated with the pine weevil. 2-Methoxyphenol, 2-phenylethanol, 3-methyl-1-butanol were found in the headspace from Rahnella aquatilis when grown in substrate containing pine bark. 2-Methoxyphenol and 3-methyl-1-butanol, phenol and methyl salicylate were found in pine feces. Birch and bilberry feces emitted mainly linalool oxides and bilberry emitted also small amounts of 2-phenylethanol.

    A second part of the thesis discusses the role of fungi in forest insect interactions and the production of oxygenated monoterpenes as possible antifeedants. Spruce bark beetles (Ips typhographus L.) aggregate with the help of pheromones and with collected forces they kill weakened adult trees as a result of associated fungi growth and larval development. A fungi associated with the bark beetle, Grosmannia europhoides, was shown to produce de novo 2-methyl-3-buten-2-ol, the major component of the spruce bark beetle aggregation pheromone. Chemical defense responses against Endoconidiophora polonica and Heterobasidion parviporum were investigated using four clones of Norway spruce with different susceptibility to Heterobasidion sp. Clone specific differences were found in induced mono-, sesqui and diterpenes. A number of oxygenated monoterpenes which are known antifeedants for the pine weevil were produced in the infested areas.

  • 5.
    Axelsson, Karolin
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Nilsson, Louise
    Nordlander, Göran
    Dep. of Ecology, SLU.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Terenius, Olle
    Dep of Ecology, SLU.
    Do pine weevil microbiota and corresponding volatiles change due to selective feeding?Manuscript (preprint) (Other academic)
  • 6.
    Axelsson, Karolin
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Zendegi-Shiraz, Amene
    Swedjemark, Gunilla
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Zhao, Tao
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Clone specific chemical defense responses in Norway spruce to infestations by two pathogenic fungi2016In: Forest Pathology, ISSN 1437-4781, E-ISSN 1439-0329Article in journal (Other academic)
    Abstract [en]

    Heterobasidion parviporum (Hp) were investigated using four clones of Norway spruce (Picea abies) with different susceptibility to Heterobasidion sp. Eight year old trees were inoculated with Ep and Hp to minimize the variation due to environment. After three weeks the bark tissue at the upper border of the inoculation hole were extracted with hexane and analyzed by GC-MS. Both treatment and clonal differences were found based on induced mono-, sesqui- and diterpenes. In addition, the Hp produced toxin, fomanoxin, was identified in lowest amount in the most Hp susceptible clone. The clonal trees seem to use different defense strategies towards the two fungi. One of the clones was able to induce strong chemical defense against both fungi, one clone induced chemical defense only against Ep and the most susceptible clone exhibited the least capacity to produce an effective defense against Ep and Hp. Two diterpenes were found to be distinctly different between clones with different susceptibilities, which can be used as chemical indication of Norway spruce resistance against fungi.

  • 7.
    Barrozo, Alexandre
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esguerra, Mauricio
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Marloie, Gael
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Florian, Jan
    Loyola Univ Chicago, Dept Chem & Biochem, Chicago, IL 60660 USA..
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Computer simulations of the catalytic mechanism of wild-type and mutant beta-phosphoglucomutase2018In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 16, no 12, p. 2060-2073Article in journal (Refereed)
    Abstract [en]

    beta-Phosphoglucomutase (beta-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of beta-glucose-1-phosphate to -glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of beta-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

  • 8.
    Berthold, Malin
    Stockholm University, Faculty of Science.
    Galanin: ligand - receptor interactions1997Doctoral thesis, comprehensive summary (Other academic)
  • 9.
    Bohman, Björn
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Unelius, C. Rikard
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Synthesis of all four stereoisomers of 5-hydroxy-4-methyl-3-heptanone using plants and oyster mushrooms2009In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 65, no 42, p. 8697-8701Article in journal (Refereed)
    Abstract [en]

    All four possible stereoisomers of 5-hydroxy-4-methyl-3-heptanone were synthesized from common achiral reagents using fast, straightforward organic synthesis, including the use of whole tissue of Daucus carota, Solanum melongena, and Pleurotus ostreatus.

  • 10. Cervin, Nicholas T.
    et al.
    Andersson, Linnea
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Ng, Jovice Boon Sing
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Olin, Pontus
    Bergström, Lennart
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Wågberg, Lars
    Lightweight and Strong Cellulose Materials Made from Aqueous Foams Stabilized by Nanofibrillated Cellulose2013In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 2, p. 503-511Article in journal (Refereed)
    Abstract [en]

    A lightweight and strong porous cellulose material has been prepared by drying aqueous foams stabilized with surface-modified nanofibrillated cellulose (NFC). This material differs from other dry, particle stabilized foams in that renewable cellulose is used as stabilizing particles. Confocal microscopy and high speed video imaging show that the octylamine-coated, rod-shaped NFC nanoparticles residing at the air-liquid interface prevent the air bubbles from collapsing or coalescing. Stable wet foams can be achieved at solids content around 1% by weight. Careful removal of the water results in a cellulose-based material with a porosity of 98% and a density of 30 mg cm(-3). These porous cellulose materials have a higher Young's modulus than porous cellulose materials made from freeze-drying, at comparable densities, and have a compressive energy absorption of 56 kJ m(-3) at 80% strain. Measurement with the aid of an autoporosimeter revealed that most pores are in the range of 300 to 500 mu m.

  • 11.
    Danielsson, Marie
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Tracing induced stress sites in conifers by single needle analysesManuscript (preprint) (Other academic)
  • 12.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Univ Oxford, Chem Res Lab, 12 Mansfield Rd, Oxford OX1 3TA, England.;Univ Oxford, Phys & Theoret Chem Lab, S Parks Rd, Oxford OX1 3QZ, England..
    Barrozo, Alexandre
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Williams, Nicholas H.
    Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England..
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 33, p. 10664-10673Article in journal (Refereed)
    Abstract [en]

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

  • 13. El-Sayed, Ashraf
    et al.
    Manning, Lee-Anne
    Unelius, C. Rikard
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Park, Kye-Chung
    Stringer, Lloyd
    White, Nicola
    Bunn, Barry
    Twidle, Andrew
    Suckling, Max
    Attraction and Antennal Response of the Common Wasp, Vespula vulgaris (L.), to Selected Synthetic Chemicals in New Zealand Beech Forests2009In: Pest Management Science, ISSN 1526-498X, E-ISSN 1526-4998, Vol. 65, no 9, p. 975-981Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The common wasp, Vespula vulgaris (L.), and the German wasp, Vespula germanica (F.), are significant problems in New Zealand beech forests (Nothofagus spp.), adversely affecting native birds and invertebrate biodiversity. This work was undertaken to develop synthetic attractants for these species to enable more efficient monitoring and management. RESULTS: Seven known wasp attractants (acetic acid, butyl butyrate, isobutanol, heptyl butyrate, octyl butyrate and 2,4-hexadienyl butyrate) were field tested, and only heptyl butyrate and octyl butyrate attracted significantly higher numbers of wasps than a non-baited trap. Accordingly, a series of straight-chain esters from methyl to decyl butyrate were prepared and field tested for attraction of social wasps. Peak biological activity occurred with hexyl butyrate, heptyl butyrate, octyl butyrate and nonyl butyrate. Polyethylene bags emitting approximately 18.4-22.6 mg day(-1) of heptyl butyrate were more attractive than polyethylene bags emitting approximately 14.7-16.8 mg day(-1) of heptyl butyrate in the field. Electroantennogram (EAG) studies indicated that queens and workers of V. vulgaris had olfactory receptor neurons responding to various aliphatic butyrates. CONCLUSION: These results are the first to be reported on the EAG response and the attraction of social wasps to synthetic chemicals in New Zealand beech forests and will enable monitoring of social wasp activity in beech forests. (C) 2009 Society of Chemical industry

  • 14.
    Ersmark, Karolina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Nervall, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hamelink, Elizabeth
    Janka, Linda K.
    Clemente, Jose C.
    Dunn, Ben M.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Samuelsson, Bertil
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Macrocyclic inhibitors of the malarial aspartic proteases plasmepsin I, II, and IV2006In: Biorganic & Medicinal Chemistry, no 14, p. 2197-2208Article in journal (Refereed)
  • 15.
    Fagerland, Jenny
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Finne-Wistrand, Anna
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Polymer Technology.
    Numata, Keiji
    Short One-Pot Chemo-Enzymatic Synthesis of L-Lysine and L-Alanine Diblock Co-Oligopeptides2014In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, no 3, p. 735-743Article in journal (Refereed)
    Abstract [en]

    Amphiphilic diblock co-oligopeptides are interesting and functional macromolecular materials for biomedical applications because of their self-assembling properties. Here, we developed a synthesis method for diblock co-oligopeptides by using chemo-enzymatic polymerization, which was a relatively short (30 min) and efficient reaction (over 40% yield). Block and random oligo(L-lysine-co-L-alanine) [oligo(Lys-co-Ala)] were synthesized using activated papain as enzymatic catalyst. The reaction time was optimized according to kinetic studies of oligo(L-alanine) and oligo(L-lysine). Using H-1 NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we confirmed that diblock and random co-oligopeptides were synthesized. Optical microscopy further revealed differences in the crystalline morphology between random and block co-oligopeptides. Plate-like, hexagonal, and hollow crystals were formed due to the strong impact of the monomer distribution and pH of the solution. The different crystalline structures open up interesting possibilities to form materials for both tissue engineering and controlled drug/gene delivery systems.

  • 16.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Conde-Alvarez, Raquel
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Holst, Otto
    Iriarte, Maite
    Zhao, Yun
    Arce-Gorvel, Vilma
    Hanniffy, Sean
    Gorvel, Jean-Pierre
    Moriyon, Ignacio
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural Studies of Lipopolysaccharide-defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 14, p. 7727-7741Article in journal (Refereed)
    Abstract [en]

    The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManB(core) proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)[beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5)]-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P), in addition to components lacking one of the terminal beta-D-GlcpN and/or the beta-D-Glcp residues (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of man-B-core gives rise to a deep-rough pentasaccharide core (beta-D-Glcp-(1 -> 4)-alpha-Kdop-(2 -> 4)-alpha-Kdop-(2 -> 6)-beta-D-GlcpN3N4P-(1 -> 6)-alpha-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal beta-D-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManB(core) proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyltransferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the beta-D-GlcpN-(1 -> 6)-beta-D-GlcpN-(1 -> 4)[beta-D-GlcpN-(1 -> 6)]-beta-D-GlcpN-(1 -> 3)-alpha-D-Manp-(1 -> 5) structure in virulence.

  • 17.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Kovacs, Helena
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    NMR structure analysis of uniformly 13C-labeled carbohydrates2014In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 59, no 2, p. 95-110Article in journal (Refereed)
    Abstract [en]

    In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of C-13-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly C-13-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-C-13)-sucrose, 342 Da] and one compound of medium molecular weight (C-13-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, similar to 10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The C-13 resonances are traced using C-13-C-13 correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the C-13 resonances, the H-1 chemical shifts are derived in a straightforward manner using one-bond H-1-C-13 correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J (CC) splitting of the C-13 resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either C-13 or H-1 detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T-2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the H-1-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the C-13-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with N-15 at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and C-13-detected (H)CACO spectra.

  • 18.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Lundborg, Magnus
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural studies and biosynthetic aspects of the o antigen polysaccharide from Escherichia coli o1742012In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 354, p. 102-105Article in journal (Refereed)
    Abstract [en]

    The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with H-1 and C-13 NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by H-1, C-13-heteronuclear multiple-bond correlation and H-1, H-1-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: -> 4)-beta-D-GlcpA-(1 -> 3)-beta-D-Galp-(1 -> 3)-beta-D-GalpNAc-(1 -> vertical bar beta-D-GlcpNAc-(1 -> 2) Cross-peaks of low intensity were present in the NMR spectra consistent with a beta-D-GlcpNAc-(1 -> 2)-beta-D-GlcpA(1 -> structural element at the terminal part of the polysaccharide, which on average is composed of similar to 15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.

  • 19.
    Ghidini, Alice
    et al.
    Karolinska Inst, Dept Biosci & Nutr, Novum, S-14183 Stockholm, Sweden..
    Bergquist, Helen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Murtola, Merita
    Karolinska Inst, Dept Biosci & Nutr, Novum, S-14183 Stockholm, Sweden.;Univ Turku, Dept Chem, Turku 20014, Finland..
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Zain, Rula
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-14186 Stockholm, Sweden.;Karolinska Univ Hosp, Ctr Rare Dis, S-17176 Stockholm, Sweden..
    Stromberg, Roger
    Karolinska Inst, Dept Biosci & Nutr, Novum, S-14183 Stockholm, Sweden..
    Clamping of RNA with PNA enables targeting of microRNA2016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 23, p. 5210-5213Article in journal (Refereed)
    Abstract [en]

    To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.

  • 20. Hederos, Sofia
    et al.
    Broo, Kerstin
    Jakobsson, Emma
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Kleywegt, Gerard J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Mannervik, Bengt
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Baltzer, Lars
    Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Biochemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-12004In: Proc. Nat. Acad. Sci., Vol. 101, p. 13163-13167Article in journal (Refereed)
    Abstract [en]

    A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.

  • 21.
    Jamroskovic, Jan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Livendahl, Madeleine
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Eriksson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sabouri, Nasim
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Identification of Compounds that Selectively Stabilize Specific G-Quadruplex Structures by Using a Thioflavin T-Displacement Assay as a Tool2016In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 22, no 52, p. 18932-18943Article in journal (Refereed)
    Abstract [en]

    Small molecules are used in the G-quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT-displacement high-throughput screening assay to find novel and selective G4-binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4-structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.

  • 22.
    Le Normand, Myriam
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology, Wood Chemistry and Pulp Technology.
    Insect repellents in spruce bark2009Conference paper (Other academic)
  • 23.
    Li, Qing
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Conformationally Constrained Oligonucleotides for RNA Targeting2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A short oligonucleotide sequence as in a single-stranded antisense oligo nucleotides (AON) or in double-stranded small interfering RNAs (siRNA) can modulate the gene expression by targeting against the cellular mRNA, which can be potentially exploited for therapeutic purposes in the treatment of different diseases. In order to improve the efficacy of oligonucleotide-based drugs, the problem of target affinity, nuclease stability and delivery needs to be addressed. Chemical modifications of oligonucleotides have been proved to be an effective strategy to counter some of these problems.

    In this thesis, chemical synthesis of conformationally constrained nucleosides such as 7′-Me-carba-LNA-A, -G, -MeC and -T as well as 6′, 7′-substituted α-L-carba-LNA-T (Papers I-III) was achieved through a key free-radical cyclization. 1D and 2D NMR techniques were employed to prove the formation of bicyclic ring system by free-radical ring closure as well as to identify the specific constrained conformations in sugar moieties. These sugar-locked nucleosides were transformed to the corresponding phosphoramidites and incorporated into antisense oligonucleotides in different sequences, to evaluate their physicochemical and biochemical properties for potential antisense-based therapeutic application.

    AONs modified with 7′-Me-carba-LNA analogues exhibited higher RNA affinities (plus 1-4°C/modification) (Papers I & III), but AONs containing α-L-carba-LNA analogues showed decreased affinities (minus 2-3°C/ modification) (Paper II) towards complementary RNA compared to the native counterpart.  It has been demonstrated in Papers I-III that 7′-methyl substitution in α-L-carba-LNA caused the Tm drop due to a steric clash of the R-configured methyl group in the major groove of the duplex, whereas 7′-methyl group of carba-LNA locating in the minor groove of the duplex exerted no obviously negative effect on Tms, regardless of its orientation. Moreover, AONs containing 7′-Me-carba-LNA and α-L-carba-LNA derivatives were found to be nucleolytically more stable than native AONs, LNA modified AONs as well as α-L-LNA modified ones (Papers I-III). We also found in Paper II & III that the orientations of OH group in C6′ of α-L-carba-LNAs and methyl group in C7′ of 7′-Me-carba-LNAs can significantly influence the nuclease stabilities of modified AONs. It was proved that the methyl substitution in cLNAs which points towards the vicinal 3′-phosphate were more resistant to nuclease degradation than that caused by the methyl group pointing away from 3′-phosphate.

    Additionally, AONs modified with 7′-Me-carba-LNAs and α-L-carba-LNAs were found to elicit the RNase H mediated RNA degradation with comparable or higher rates (from 2-fold to 8-fold higher dependent upon the modification sites) as compared to the native counterpart. We also found that the cleavage patterns and rates by E. coli RNase H1 were highly dependent upon the modification sites in the AON sequences, regardless of the structural features of modifications (Papers II & III). Furthermore, we have shown that the modulations of Tms of AON/RNA duplexes are directly correlated with the aqueous solvation (Paper III).

  • 24.
    Liao, Rong-Zhen
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry. Huazhong University of Science & Technology, People's Republic of China.
    Siegbahn, Per E. M.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Mechanism for O-O bond formation in a biomimetic tetranuclear manganese cluster - A density functional theory study2015In: Journal of Photochemistry and Photobiology. B: Biology, ISSN 1011-1344, E-ISSN 1873-2682, Vol. 152, no Part A, p. 162-172Article in journal (Refereed)
    Abstract [en]

    Density functional theory calculations have been used to study the reaction mechanism of water oxidation catalyzed by a tetranuclear Mn-oxo cluster Mn4O4L6 (L = (C6H4)(2)PO4-). It is proposed that the O-O bond formation mechanism is different in the gas phase and in a water solution. In the gas phase, upon phosphate ligand dissociation triggered by light absorption, the O-O bond formation starting with both the Mn-4(III,III,IV,IV) and Mn-4(III,IV,IV,IV) oxidation states has to take place via direct coupling of two bridging oxo groups. The calculated barriers are 42.3 and 37.1 kcal/mol, respectively, and there is an endergonicity of more than 10 kcal/mol. Additional photons are needed to overcome these large barriers. In water solution, water binding to the two vacant sites of the Mn ions, again after phosphate dissociation triggered by light absorption, is thermodynamically and kinetically very favorable. The catalytic cycle is suggested to start from the Mn-4(III,III,III,IV) oxidation state. The removal of three electrons and three protons leads to the formation of a Mn-4(III,IV,IV,IV)-oxyl radical complex. The O-O bond formation then proceeds via a nucleophilic attack of water on the Mn-IV-oxyl radical assisted by a Mn-bound hydroxide that abstracts a proton during the attack. This step was calculated to be rate-limiting with a total barrier of 29.2 kcal/mol. This is followed by proton-coupled electron transfer, O-2 release, and water binding to start the next catalytic cycle.

  • 25.
    Liu, Jun
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials. Jiangsu Univ, Sch Environm & Safety Engn, Biofuels Inst, Zhenjiang.
    Bacher, Markus
    Univ Nat Resources & Appl Life Sci BOKU, Dept Chem, Vienna.
    Rosenau, Thomas
    Univ Nat Resources & Appl Life Sci BOKU, Dept Chem, Vienna; Åbo Akad Univ, Lab Wood & Paper Chem, Johan Gadolin Proc Chem Ctr, Turku.
    Willfoer, Stefan
    Åbo Akad Univ, Lab Wood & Paper Chem, Johan Gadolin Proc Chem Ctr, Turku.
    Mihranyan, Albert
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Nanotechnology and Functional Materials.
    Potentially Immunogenic Contaminants in Wood-Based and Bacterial Nanocellulose: Assessment of Endotoxin and (1,3)-β-d-Glucan Levels2018In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, no 1, p. 150-157Article in journal (Refereed)
    Abstract [en]

    Knowledge gaps in the biosafety data of the nanocellulose (NC) for biomedical use through various routes of administration call for closer look at health and exposure evaluation. This work evaluated the potentially immunogenic contaminants levels, for example, endotoxin and (1,3)-β-d-glucan, in four representative NCs, that is, wood-based NCs and bacterial cellulose (BC). The hot-water extracts were analyzed with ELISA assays, HPSEC-MALLS, GC, and NMR analysis. Varying levels of endotoxin and (1,3)-β-d-glucan contaminats were found in these widely used NCs. Although the β-(1,3)-d-glucan was not detected from the NMR spectra due to the small extract samples amount (2–7 mg), the anomerics and highly diastereotopic 6-CH2 signals may suggest the presence of β-(1,4)-linkages with β-(1,6) branching in the polysaccharides of NCs’ hot-water extracts, which were otherwise not detectable in the enzymatic assay. In all, the article highlights the importance of monitoring various water-soluble potentially immunogenic contaminants in NC for biomedical use.

  • 26.
    Lundborg, Lina
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Effects of methyl jasmonate on chemical defenses of conifer seedlings in relation to feeding by Hylobius abietis2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The chemical elicitor methyl jasmonate (MeJA) could replace insecticides in Europe and Asia for protection of young conifers against the pine weevil (Hylobius abietis). This thesis mainly focuses on the effects of MeJA treatment on chemical defenses of conifers from seedling batches with documented field resistance. Tissues of three pine species and one spruce species, with various treatments, were here extracted in hexane, whereafter volatile contents of tissues were separated and analyzed by 2D GC-MS.

    Induced responses of seedlings of Maritime pine (Pinus pinaster) and Monterey pine (Pinus radiata) to the folivore pine processionary (Thaumetopoea pityocampa), and the phloem-feeder H. abietis, have been studied. Amounts of mono- and sesqui-terpenes (and also non-volatile resin) in conifer tissues (needles and phloem) were less induced by T. pityocampa than by H. abietis.

    The MeJA-treated seedlings of Scots pine (Pinus sylvestris) changed their composition of phloem monoterpenes (induction of (-)-β-pinene), and were better protected in field than the seedlings of Norway spruce (Picea abies), which increased their total amounts of monoterpenes. Orientation bioassays with H. abietis showed deterrent effects of (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate and 1,8-cineole. Conversely, (-)-α-pinene (induced in P. abies but not in P. sylvestris) was non-deterrent.

    MeJA-treated seedlings fed on by H. abietis contained higher amounts of the H. abietis antifeedant 2-phenylethanol, in tissues and emissions. Phloem of control seedlings instead induced the (+)-α-pinene, which is one of the enantiomers of the H. abietis attractant α-pinene.

    In response to MeJA treatment, the relative amounts of the (+)-α-pinene increased in the phloem of  P. radiata, while it decreased in P. pinaster phloem. The preference of H. abietis in the field for P. radiata before P. pinaster may be explained by these changes in enantiomers of the H. abietis attractant α-pinene.

  • 27.
    Lundborg, Lina
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Fedderwitz, Frauke
    Department of Ecology, Swedish University of Agricultural Sciences.
    Björklund, Niklas
    Department of Ecology, Swedish University of Agricultural Sciences.
    Nordlander, Göran
    Department of Ecology, Swedish University of Agricultural Sciences.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Conifer chemical defenses influence meal properties of the pine weevil Hylobius abietisManuscript (preprint) (Other academic)
    Abstract [en]

    Chemical defenses of conifers can be experimentally activated to respond more efficiently to feeding damage by insects. One chemical elicitor that triggers plant defenses, and thereby protects conifers, is methyl jasmonate (MeJA). However, there is little known about the associations between MeJA-induced conifer defenses, and the meal properties of phytophagous insects. To address this knowledge gap, we have analyzed relations between volatile contents of Scots pine (Pinus sylvestris L.) tissues, and meal properties of the pine weevil (Hylobius abietis (L.)). Phloem and needles (both weevil target tissues) from MeJA-treated and control seedlings were extracted by n-hexane and analyzed by two-dimensional gas chromatography-mass spectrometry (2D GC-MS). Feeding of pine weevils on seedlings from the same batches were video-recorded to determine meal properties. Multivariate statistical analyses showed that phloem and needle contents of MeJA-treated seedlings had different volatile compositions from the control seedlings. Levels of (+)-α-pinene were high, and levels of the antifeedant 2-phenylethanol were low, in phloem of control seedlings with feeding damage. Accordingly, pine weevils fed more slowly and had shorter meals on MeJA-treated seedlings. In addition, the chemical compositions of phloem and needles – and accordingly weevil meal durations on them – were more similar in MeJA-treated seedlings than in controls. The results illustrate that detailed knowledge of insect responses can help efforts to identify and elucidate specific roles of resistance agents in complex chemical profiles.

  • 28.
    Lundborg, Lina
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Fedderwitz, Frauke
    Department of Ecology, Swedish University of Agricultural Sciences.
    Björklund, Niklas
    Department of Ecology, Swedish University of Agricultural Sciences.
    Nordlander, Göran
    Department of Ecology, Swedish University of Agricultural Sciences.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Induced defenses change the chemical composition of pine seedlings and influence meal properties of the pine weevil Hylobius abietis2016In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 130, p. 99-105Article in journal (Refereed)
    Abstract [en]

    The defense of conifers against phytophagous insects relies to a large extent on induced chemical defenses. However, it is not clear how induced changes in chemical composition influence the meal properties of phytophagous insects (and thus damage rates). The defense can be induced experimentally with methyl jasmonate (MeJA), which is a substance that is produced naturally when a plant is attacked. Here we used MeJA to investigate how the volatile contents of Scots pine (Pinus sylvestris L.) tissues influence the meal properties of the pine weevil (Hylobius abietis (L.)). Phloem and needles (both weevil target tissues) from MeJA-treated and control seedlings were extracted by n-hexane and analyzed by two-dimensional gas chromatography-mass spectrometry (2D GC-MS). The feeding of pine weevils on MeJA-treated and control seedlings were video-recorded to determine meal properties. Multivariate statistical analyses showed that phloem and needle contents of MeJA-treated seedlings had different volatile compositions compared to control seedlings. Levels of the pine weevil attractant (+)-α-pinene were particularly high in phloem of control seedlings with feeding damage. The antifeedant substance 2-phenylethanol occurred at higher levels in the phloem of MeJA-treated than in control seedlings. Accordingly, pine weevils fed slower and had shorter meals on MeJA-seedlings. The chemical compositions of phloem and needle tissues were clearly different in control seedlings but not in the MeJA-treated seedlings. Consequently, meal durations of mixed meals, i.e. both needles and phloem, were longer than phloem meals on control seedlings, while meal durations on MeJA seedlings did not differ between these meal contents. The meal duration influences the risk of girdling and plant death. Thus our results suggest a mechanism by which MeJA treatment may protect conifer seedlings against pine weevils.

  • 29.
    Lundborg, Lina
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Nordlander, Göran
    Department of Ecology, Swedish University of Agricultural Sciences.
    Björklund, Niklas
    Department of Ecology, Swedish University of Agric.
    Nordenhem, Henrik
    Department of Ecology, Swedish University of Agric.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Methyl jasmonate-induced monoterpenes in Scots pine and Norway spruce tissues affect pine weevil orientationManuscript (preprint) (Other academic)
    Abstract [en]

    In large parts of Europe insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least partly due to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (-)-β-pinene in phloem (the weevil’s main target tissue) of both tree species, however, the (-)-β-pinene/(-)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (-)-β-pinene, (+)-3-carene, (-)-bornyl acetate or 1,8-cineole. However, (-)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (-)-β-pinene, in contrast to strong increases in both non-deterrent (-)-α-pinene and the deterrent (-)-β-pinene in P. abies seedlings.

  • 30.
    Lundborg, Lina
    et al.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Sampedro, Luis
    Grupo de Xenetica e Ecoloxía da Resistencia en Coníferas, Misión Biológica de Galicia (MBG-CSIC), Pontevedra, Spain.
    Zas, Rafael
    Grupo de Xenetica e Ecoloxía da Resistencia en Coníferas, Misión Biológica de Galicia (MBG-CSIC), Pontevedra, Spain.
    Borg-Karlson, Anna-Karin
    KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
    Constitutive and MeJA-induced terpenes in Pinus pinaster and Pinus radiata in relation to Hylobius abietisManuscript (preprint) (Other academic)
    Abstract [en]

    The feeding preference of the pine weevil to Maritime pine (Pinus pinaster Ait.) and Monterey pine (Pinus radiata D. Don) differ in laboratory tests and in the field: Can this be explained by the volatile compounds released by the seedlings? Understanding the interaction between the chemical defenses of young pines and feeding preferences of the pine weevil (Hylobius abietis (L.)) may improve protection methods of conifer seedlings in Europe and Asia. In order to increase the resistance of seedlings of P. pinaster and P. radiata, these were treated with the chemical elicitor methyl jasmonate (MeJA). The effects of the MeJA treatments on the amounts and composition of volatile terpenes in tissues (needles and phloem) were investigated by extracting these in hexane, whereafter the volatile constituents of the extracts were separated and identified by gas chromatography-mass spectrometry (GC-MS). In addition, the most abundant monoterpenes (α-pinene and β-pinene) were separated on a chiral column in their enantiomers. Already at the lowest concentration of MeJA treatment, 5 mM, the amounts of volatile terpenes in the needles of P. radiata increased and the seedling heights were reduced. In P. pinaster, on the other hand, no effect from the MeJA treatment was observed on the total volatile terpenes in the needles, and only at the strongest MeJA concentration, the seedling heights were affected. In the phloem of P. pinaster the composition of volatile contents, changed after MeJA treatment, showing a reduction in the (+)-α-pinene. In contrast, the amounts of (+)-α-pinene increased in P. radiata. In a previous study, controls and MeJA-treated pine seedlings, from the same MeJA treatment batches as in this study, were subjected to a field trial. In the field, the pine weevils preferred the seedlings of P. radiata over those of P. pinaster. We propose that the differences in induction of (+)-α-pinene may be important for explaining the differences in pine weevil feeding preferences.

  • 31.
    Lundborg, Magnus
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy2011In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 11, p. 3851-3855Article in journal (Refereed)
    Abstract [en]

    The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (-)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic 1H and 13C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user's point of view only the unknown sample has to be derivatized for use in CASPER.

  • 32. Mally, Manuela
    et al.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    LeibundGut-Landmann, Salome
    Laacisse, Lamia
    Fan, Yao-Yun
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Aebi, Markus
    Glycoengineering of host mimicking type-2 LacNAc polymersand Lewis X antigens on bacterial cell surfaces2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 87, no 1, p. 112-131Article in journal (Refereed)
    Abstract [en]

    Bacterial carbohydrate structures play a central role in mediating a variety of host-pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram-negative bacteria, is composed of a lipid A-core and the O-antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Gal beta 1-4(Fuc alpha 1-3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram-negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O-antigen. A ubiquitous building block in mammalian N-linked protein glycans is Gal beta 1-4GlcNAc, referred to as a type-2 N-acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A-cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly-LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI-TOF and NMR analysis. Glycoengineered LOS induced pro-inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.

  • 33.
    Marshall, D. G.
    et al.
    AgResearch Ltd, New Zealand.
    Jackson, T. A.
    AgResearch Ltd, New Zealand.
    Unelius, C. Rikard
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. New Zealand Inst Plant & Food Res Ltd, New Zealand.
    Wee, S. L.
    New Zealand Inst Plant & Food Res Ltd, New Zealand ; Univ Kebangsaan Malaysia, Malaysia.
    Young, S. D.
    AgResearch Ltd, New Zealand.
    Townsend, R. J.
    AgResearch Ltd, New Zealand.
    Suckling, D. M.
    New Zealand Inst Plant & Food Res Ltd, New Zealand ; Univ Auckland, New Zealand.
    Morganella morganii bacteria produces phenol as the sex pheromone of the New Zealand grass grub from tyrosine in the colleterial gland2016In: The Science of Nature: Naturwissenschaften, ISSN 0028-1042, E-ISSN 1432-1904, Vol. 103, no 7-8, article id 59Article in journal (Refereed)
    Abstract [en]

    Costelytra zealandica (Coleoptera: Scarabeidae) is a univoltine endemic species that has colonised and become a major pest of introduced clover and ryegrass pastures that form about half of the land area of New Zealand. Female beetles were previously shown to use phenol as their sex pheromone produced by symbiotic bacteria in the accessory or colleterial gland. In this study, production of phenol was confirmed from the female beetles, while bacteria were isolated from the gland and tested for attractiveness towards grass grub males in traps in the field. The phenol-producing bacterial taxon was identified by partial sequencing of the 16SrRNA gene, as Morganella morganii. We then tested the hypothesis that the phenol sex pheromone is biosynthesized from the amino acid tyrosine by the bacteria. This was shown to be correct, by addition of isotopically labelled tyrosine (C-13) to the bacterial broth, followed by detection of the labelled phenol by SPME-GCMS. Elucidation of this pathway provides specific evidence how the phenol is produced as an insect sex pheromone by a mutualistic bacteria.

  • 34.
    Mindemark, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
    Functional Cyclic Carbonate Monomers and Polycarbonates: Synthesis and Biomaterials Applications2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The present work describes a selection of strategies for the synthesis of functional aliphatic polycarbonates. Using an end-group functionalization strategy, a series of DNA-binding cationic poly(trimethylene carbonate)s was synthesized for application as vectors for non-viral gene delivery. As the end-group functionality was identical in all polymers, the differences observed in DNA binding and in vitro transfection studies were directly related to the length of the hydrophobic poly(trimethylene carbonate) backbone and the number of functional end-groups. This enabled the use of this polymer system to explore the effects of structural elements on the gene delivery ability of cationic polymers, revealing striking differences between different materials, related to functionality and cationic charge density.

    In an effort to achieve more flexibility in the synthesis of functional polymers, polycarbonates were synthesized in which the functionalities were distributed along the polymer backbone. Through polymerization of a series of alkyl halide-functional six-membered cyclic carbonates, semicrystalline chloro- and bromo-functional homopolycarbonates were obtained. The tendency of the materials to form crystallites was related to the presence of alkyl as well as halide functionalities and ranged from polymers that crystallized from the melt to materials that only crystallized on precipitation from a solution. Semicrystallinity was also observed for random 1:1 copolymers of some of the monomers with trimethylene carbonate, suggesting a remarkable ability of repeating units originating from these monomers to form crystallites.

    For the further synthesis of functional monomers and polymers, azide-functional cyclic carbonates were synthesized from the bromo-functional monomers. These were used as starting materials for the click synthesis of triazole-functional cyclic carbonate monomers through Cu(I)-catalyzed azide–alkyne cycloaddition. The click chemistry strategy proved to be a viable route to obtain structurally diverse monomers starting from a few azide-functional precursors. This paves the way for facile synthesis of a wide range of novel functional cyclic carbonate monomers and polycarbonates, limited only by the availability of suitable functional alkynes.

  • 35.
    Morin, Dominique
    et al.
    BRGM, Bureau de Recherches Géologiques et Minières.
    Lips, Andor
    BRGM, Bureau de Recherches Géologiques et Minières.
    Pinches, Tony
    Mintek, Randburg, South Africa.
    Huisman, Jacco
    Paques, El Balk, The Netherlands.
    Frías Gomez, Carlos
    Técnicas Reunidas, Madrid, Spain.
    Norberg, Anders
    Skeria, Skellefteå.
    Eric, Forssberg
    Luleå tekniska universitet.
    BioMinE: integrated project for the development of biotechnology for metal-bearing materials in Europe2006In: Hydrometallurgy, ISSN 0304-386X, E-ISSN 1879-1158, Vol. 83, no 1-4, p. 69-76Article in journal (Refereed)
    Abstract [en]

    Biohydrometallurgy is the offspring of the unexpected union of biotechnology and metallurgy. From specific properties of some extreme biotopes, active principles of interactions between microbial metabolisms and minerals have been extracted to be used as efficient metallurgical processes.

    Many profitable industrial operations based on these bioprocesses have been running to recover copper, gold, uranium or cobalt for instance and many other applications have been designed.

    Europe was quite active in this area in the past, but currently the leadership is in South Africa, America and Australia.

    BioMinE (Biotechnology for Metal-bearing material In Europe) is a large integrated project launched with the support of the European Commission. It is aimed at stimulating synergies between the most relevant universities, research and industrial organisations to develop new concepts in this technical field that allow a better exploitation of the mineral resources in the future.

    The main technical subject is the investigation of the opportunities to apply bioleach processes to primary and secondary resources of metal-bearing materials. The second technical area of the project in terms of effort is the study of the recovery of metals from pregnant bioleach solution using biological reagents. All along the project duration, these investigations are focussed on the relevant resources in Europe screened according to an iterative process. The integration of the innovative pathways of processing will be evaluated up to the pilot scale whenever it is appropriate.

    The Consortium of BioMinE comprises 35 partners from industry (12 including 5 SMEs) research organisations (9) universities (14) and government (2). The participants are from 12 EU member states, from 1 candidate country (Romania), and from South Africa (INCO Country).

    The overall budget of the project is 17.9 million Euros, with a contribution from the European Commission of 11.6 million Euros. Started on November 1, 2004, the project will last 4 years.

    An overview of BioMinE in the general context of the biohydrometallurgy development is the subject of this presentation.

  • 36.
    Morin, Dominique
    et al.
    BRGM, Bureau de Recherches Géologiques et Minières.
    Pinches, Tony
    Mintek, Randburg, South Africa.
    Huisman, Jacco
    Paques, AB Balk, The Netherlands.
    Frías, Carlos
    Técnicas Reunidas, Madrid, Spain.
    Norberg, Anders
    Skeria, Skellefteå.
    Forssberg, Eric
    Luleå tekniska universitet.
    Progress after three years of BioMinE-Research and Technological Development project for a global assessment of biohydrometallurgical processes applied to European non-ferrous metal resources2008In: Hydrometallurgy, ISSN 0304-386X, E-ISSN 1879-1158, Vol. 94, no 1-4, p. 58-68Article in journal (Refereed)
    Abstract [en]

    BioMinE is an integrated project under the sixth framework programme of research supported by the European Commission, which started in November 2004 and will last until October 2008 (Ref. NMP2-CT-2005-500329). It is dedicated to the evaluation of biohydrometallurgy to improve the exploitation of the European non-ferrous metal resources in a sustainable way. At the end of 2007, the Consortium of BioMinE comprised 37 partners from industry (13 including 6 Small or Medium Enterprises), research organisations (8), universities (15), and government (1). The participants are from 13 EU member states and from Serbia and South Africa (INCO Countries). For more details see http://biomine.brgm.fr.

    The three main kinds of resources considered for bioleaching studies are:

    - Copper polymetallics (concentrates and tailings),

    - Zinc polymetallics (zinc and zinc polymetallic concentrates)

    - Secondary wastes (tailings, rock and metallurgical wastes, etc.)

    For each of these resources, amenability studies of application of bioleaching technologies by various approaches have been undertaken or still ongoing. Further processing assessment will be conducted up to the demonstration scale. Technological improvements have been made to apply bioleaching in the context of the European resources in terms of complexity and sustainability requirements. The relevant fundamental studies covering bio-prospecting, molecular ecology, biochemistry, and genetics areas aimed at improving the understanding and the control of the selected technologies have given original results.

    Much progress has also been obtained in the use of the microbial sulfate-reducing process to polish effluents and to recover metals from leachates containing low concentrations of metals. The finding of micro-organisms thriving at low and high temperature, respectively 8 and 65 °C, leads to an extension of the application range of the process. It has been also observed that this process could be pushed down to pH 4.5 and 4 creating opportunities of selective metal recovery as metal sulphides. It has also been demonstrated that sulphate can be removed at high concentrations, as well as arsenic or selenium. The next step in this work is pilot testing. This will allow to determine scale-up criteria and to assess the residual metal concentration under actual conditions.

    The pilot-scale demonstration operations, as well as the techno-economic and comparative sustainability assessments will be achieved during 2008, the last year of the project.

    The prototypes of the learning objects for training about biohydrometallurgy accessible by internet have been elaborated. A public output of this work is accessible at http://wiki.biomine.skelleftea.se/wiki. The basic knowledge thus delivered is aimed at disseminating the understanding of the origins and use of biohydrometallurgy.

    Contacts with mining operators in Europe have been taken and collaboration schemes have been established in various ways according to the respective contexts. When a high potential of technical involvement could be foreseen, a direct participation of the mining operators in the project was favoured, this led to integrate KGHM (Pol), Boliden (Sw) and Copper Institute of Bor (Serbia) into the consortium of partners.

    When no direct technical commitment was conceivable at the first stage, collaboration was established with companies with the most urgent requirement to have access to the relevant resource.

  • 37.
    Ochtrop, Philipp
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Selective protein functionalisation via enzymatic phosphocholination2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection.

  • 38.
    Ochtrop, Philipp
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ernst, Stefan
    Itzen, Aymelt
    Hedberg, Christian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Exploring the substrate scope of the phosphocholine transferase AnkX for versatile protein functionalisationManuscript (preprint) (Other academic)
  • 39. Olsen, E. K.
    et al.
    Hansen, E.
    Moodie, L. W. K.
    Isaksson, J.
    Sepčić, K.
    Cergolj, M.
    Svenson, Johan
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Kemi Material och Ytor, Medicinteknik.
    Andersen, J. H.
    Marine AChE inhibitors isolated from Geodia barretti: Natural compounds and their synthetic analogs2016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 5, p. 1629-1640Article in journal (Refereed)
    Abstract [en]

    Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 μM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

  • 40.
    Orrling, Kristina M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    On the Versatility of Microwave-Assisted Chemistry: Exemplified by Applications in Medicinal Chemistry, Heterocyclic Chemistry and Biochemistry2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics.

    In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases.

    Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes.

    In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation.

  • 41.
    Park, Sungkyu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Cyclotides evolve: Studies on their natural distribution, structural diversity, and activity2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The cyclotides are a family of naturally occurring peptides characterized by cyclic cystine knot (CCK) structural motif, which comprises a cyclic head-to-tail backbone featuring six conserved cysteine residues that form three disulfide bonds. This unique structural motif makes cyclotides exceptionally resistant to chemical, thermal and enzymatic degradation. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects.

    The cyclotides found in plants exhibit considerable sequence and structural diversity, which can be linked to their evolutionary history and that of their host plants. To clarify the evolutionary link between sequence diversity and the distribution of individual cyclotides across the genus Viola, selected known cyclotides were classified using signature sequences within their precursor proteins. By mapping the classified sequences onto the phylogenetic system of Viola, we traced the flow of cyclotide genes over evolutionary history and were able to estimate the prevalence of cyclotides in this genus. In addition, the structural diversity of the cyclotides was related to specific features of the sequences of their precursor proteins, their evolutionary selection and expression levels.

    A number of studies have suggested that the biological activities of the cyclotides are due to their ability to interact with and disrupt biological membranes. To better explain this behavior, quantitative structure-activity relationship (QSAR) models were developed to link the cyclotides’ biological activities to the membrane-interactive physicochemical properties of their molecular surfaces. Both scalar quantities (such as molecular surface areas) and moments (such as the distributions of specific properties over the molecular surface) were systematically taken into account in the development of these models. This approach allows the physicochemical properties of cyclotides to be geometrically interpreted, facilitating the development of guidelines for drug design using cyclotide scaffolds.

    Finally, an optimized microwave-assisted Fmoc-SPSS procedure for the total synthesis of cyclotides was developed. Microwave irradiation is used to accelerate and improve all the key steps in cyclotide synthesis, including the assembly of the peptide backbone by Fmoc-SPPS, the cleavage of the protected peptide, and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. 

  • 42. Perepelov, Andrei V.
    et al.
    Wang, Quan
    Filatov, Andrei V.
    Xia, Xianghong
    Shashkov, Alexander S.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Wang, Lei
    Knirel, Yuriy A.
    Structures and gene clusters of the closely related O-antigens of Escherichia coli O46 and O134, both containing D-glucuronoyl-D-allothreonine2015In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 409, p. 20-24Article in journal (Refereed)
    Abstract [en]

    The O-polysaccharides (O-antigens) were isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O46 and O134. The structures of their linear tetrasaccharide repeating units were established by sugar analysis along with 1D and 2D H-1 and C-13 NMR spectroscopy: [GRAPHICS] where D-aThr indicates D-allothreonine and R indicates O-acetyl substitution (similar to 70% on aThr and similar to 15% on GalNAc) in E. coli O46 whereas the O-acetylation is absent in E. coli O134. Functions of genes in the essentially identical O-antigen gene clusters of E. coli O46 and O134 were tentatively assigned by a comparison with sequences in available databases and found to be in agreement with the O-polysaccharide structures established.

  • 43.
    Peuker, Sebastian
    et al.
    University of Gothenburg, Gothenburg, Sweden.
    Andersson, Hanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Gustavsson, Emil
    University of Gothenburg, Gothenburg, Sweden.
    Maiti, Kiran Sankar
    University of Gothenburg, Gothenburg, Sweden.
    Kania, Rafal
    University of Gothenburg, Gothenburg, Sweden.
    Karim, Alavi
    University of Gothenburg, Gothenburg, Sweden.
    Niebling, Stephan
    University of Gothenburg, Gothenburg, Sweden.
    Pedersen, Anders
    Swedish NMR Centre at the University of Gothenburg, Gothenburg, Sweden.
    Erdelyi, Mate
    University of Gothenburg, Gothenburg, Sweden.
    Westenhoff, Sebastian
    University of Gothenburg, Gothenburg, Sweden.
    Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 7, p. 2312-2318Article in journal (Refereed)
    Abstract [en]

    Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.

  • 44.
    Popović-Bijelić, Ana
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kowol, Christian R.
    Lind, Maria E. S.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Luo, Jinghui
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Himo, Fahmi
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Enyedy, Éva A.
    Arion, Vladimir B.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study2011In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, Vol. 105, no 11, p. 1422-1431Article in journal (Refereed)
    Abstract [en]

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.

  • 45.
    Rickaby, Rosalind E. M.
    et al.
    Dept. of Earth Sciences, Oxford University, UK.
    Henderiks, Jorijntje
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology.
    Young, Jodi N.
    Perturbing phytoplankton: response and isotopic fractionation with changing carbonate chemistry in two coccolithophore species2010In: Climate of the Past, ISSN 1814-9324, Vol. 6, p. 771-785Article in journal (Refereed)
    Abstract [en]

    All species of coccolithophore appear to respond to perturbations of carbonate chemistry in a different way. Here, we show that the degree of malformation, growthrate and stable isotopic composition of organic matter and carbonate produced by two contrasting species of coccolithophore (Gephyrocapsa oceanica and Coccolithus pelagicusssp. braarudii) are indicative of differences between their photosynthetic and calcification response to changing DIC levels (ranging from 1100 to 7800 μmol kg−1) at constant pH (8.13±0.02). Gephyrocapsa oceanica thrived under all conditions of DIC, showing evidence of increased growth rates at higher DIC, but C. braarudii was detrimentally affected at high DIC showing signs of malformation, and decreased growth rates. The carbon isotopic fractionation into organic matter and the coccoliths suggests that C. braarudii utilises a common internal pool of carbon for calcification and photosynthesis but G. oceanica relies on independent supplies for each process. All coccolithophores appear to utilize bicarbonate as their ultimate source of carbon for calcification resulting in the release of a proton. But, we suggest that this proton can be harnessed to enhance the supply of CO2(aq) for photosynthesis either from a large internal HCO−3 pool which acts as a pH buffer (C. braarudii), or pumped externally to aid the diffusive supply of CO2 across the membrane from the abundant HCO−3 (G. oceanica), likely mediated by an internal and external carbonic anhydrase respectively. Our simplified hypothetical spectrum of physiologies may provide a context to understand different species response to changing pH and DIC, the species specific Ep and calcite “vital effects”, as well as accounting for geological trends in coccolithophore cell size.

  • 46.
    Rotili, Dante
    et al.
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Samuele, Alberta
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Tarantino, Domenico
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Ragno, Rino
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Musmuca, Ira
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Ballante, Flavio
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Botta, Giorgia
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Morera, Ludovica
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Pierini, Marco
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Cirilli, Roberto
    Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
    Nawrozkij, Maxim B
    Volgograd State Technical University, prospekt Lenina, 28, 400131 Volgograd, Russia.
    Gonzalez, Emmanuel
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Clotet, Bonaventura
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Artico, Marino
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    Esté, José A
    Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.
    Maga, Giovanni
    Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy.
    Mai, Antonello
    Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
    2-(Alkyl/aryl)amino-6-benzylpyrimidin-4(3H)-ones as inhibitors of wild-type and mutant HIV-1: enantioselectivity studies.2012In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, no 7, p. 3558-62Article in journal (Refereed)
    Abstract [en]

    The single enantiomers of two pyrimidine-based HIV-1 non-nucleoside reverse transcriptase inhibitors, 1 (MC1501) and 2 (MC2082), were tested in both cellular and enzyme assays. In general, the R forms were more potent than their S counterparts and racemates and (R)-2 was more efficient than (R)-1 and the reference compounds, with some exceptions. Interestingly, (R)-2 displayed a faster binding to K103N RT with respect to WT RT, while (R)-1 showed the opposite behavior.

  • 47.
    Runquist, Mårten
    Stockholm University, Faculty of Science.
    Distribution and regulation of enzymes involved in polyisoprenoid biosynthesis1995Doctoral thesis, comprehensive summary (Other academic)
  • 48.
    Saleeb, Michael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mojica, Sergio
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Eriksson, Anna U
    Andersson, C David
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Elofsson, Mikael
    Natural product inspired library synthesis - Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis.2017In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 143, p. 1077-1089, article id S0223-5234(17)31002-4Article in journal (Refereed)
    Abstract [en]

    A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial activity in a panel of test systems including phenotypic, biochemical and image-based screening assays. We identified several potent inhibitors that block intracellular replication of pathogenic Chlamydia trachomatis with IC50 ≤ 3 μM. These new C. trachomatis inhibitors can serve as starting points for the development of specific treatments that reduces the global burden of C. trachomatis infections.

  • 49.
    Sane, Prafullachandra Vishnu
    et al.
    Jain Irrigation Systems Limited, Jalgaon, India.
    Ivanov, Alexander G.
    Department of Biology and The Biotron, Experimental Climate Change Research Centre, University of Western Ontario, London, Canada.
    Öquist, Gunnar
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Huener, Norman P. A.
    Department of Biology and The Biotron, Experimental Climate Change Research Centre, University of Western Ontario, London, Canada.
    Thermoluminescence2012In: Photosynthesis: Plastid Biology, Energy Conversion and Carbon Assimilation / [ed] Julian J. Eaton-Rye,Baishnab C. Tripathy, Thomas D. Sharkey, Springer Netherlands, 2012, p. 445-474Chapter in book (Refereed)
    Abstract [en]

    Thermo luminescence (TL) of photosynthetic membranes was discovered by William Arnold and Helen Sherwood in 1957. In the last half century, several studies have elucidated the mechanism of TL emission, which showed that the recombination of different charge pairs generated and trapped during pre-illumination are responsible for the observed light emission. Since most of the TL bands originate within Photosystem II (PS II), the technique of TL has become a useful complementary tool to chlorophyll a fluorescence to probe subtle changes in PS II photochemistry. The technique is simple and non-invasive; it has been successfully used to study leaf, cells, thylakoids and even reaction center preparations. The TL technique provides quick information about the redox potential changes of the bound primary quinone (Q(A)) and the secondary quinone (Q(B)) acceptors of PS II; TL has been extensively used to study the effects of photoinhibition, mutations, stresses and myriad responses of the photosynthetic apparatus during acclimation and adaptation. This chapter reviews crucial evidence for the identification of charge pairs responsible for the generation of different TL bands; the relationship of these bands to the components of delayed light emission; responses to excitation pressure arising out of environmental factors; methodology, and instrumentation. A model based on the detailed analysis of the redox shifts of the PS II electron acceptors Q(A) and Q(B), explaining the possibility of non-radiative dissipation of excess light energy within the reaction center of PS II (reaction center quenching) and its physiological significance in photoprotection of the photosynthetic membranes has been suggested. Developments in the analysis of biophysical parameters and the non-adherence of photosynthetic TL to the analysis by the 1945 theory of J.T. Randall and M.H.F. Wilkins have been briefly reviewed.

  • 50.
    Shokeer, Abeer
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glutathione transferases (GSTs) play a pivotal role in cellular defense, since they are main contributors to the inactivation of genotoxic compounds of exogenous and endogenous origins. Directed evolution was used to improve the catalytic activities of Theta class GST T1-1 toward different substrates. The library was constructed by recombination of cDNA coding for human GST T1-1 and rodent Theta class GSTs, resulting in the F2-F5 generations. The clones were heterologously expressed in Escherichia coli and screened for variants with enhanced alkyltransferase activity. A mutant, F2:1215, with a 70-fold increased catalytic efficiency with 4-nitrophenethyl bromide (NPB) compared to human GST T1-1, was isolated from the second generation. NPB was used as a surrogate substrate of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in order to facilitate the screening process. The catalytic efficiency of the F2:1215 with BCNU had improved 170-fold compared to wild-type human GST T1-1, suggesting that NPB is a suitable model substrate for the anticancer drug BCNU. The sequence of the F2:1215 mutant differs from wild-type human GST T1-1 by three residues; one of these differences is Arg234, which corresponds to Trp in the human enzyme. Upon replacing the Trp234 in the human GST T1-1 with Arg, the resulting mutant (hTrp234Arg) showed enhanced alkyltransferase activity with a wide range of substrates (e.g. haloalkanes and other typical GSTs substrates). The three-dimensional structures of both wild-type human GST T1-1 and hTrp234Arg mutant help to explain the higher activity showed by of hTrp234Arg mutant compared to wild-type enzyme. The reciprocal mutation of the residue 234 in mouse GST T1-1 to that found in human, mArg234Trp, caused a dramatic decrease in the activity of the mouse enzyme to be similar to human GST T1-1. This indicates that residue 234 can be considered as a master switch of activities between human and rodent GST T1-1. Another important residue in the C-terminal helix of GST T1-1 is Met232. Although residue 232 points away from the H-site, it influences the catalytic activity and substrate selectivity of the mouse GST T1-1. A minor modification of Met232 induces major changes in the substrate-activity profile of the mouse GST T1-1 to favor novel substrates such as isothiocyanates and hydroperoxides and decreases the activity toward substrates that catalyzed by the wild-type enzyme.

     

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