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  • 151.
    de La Torre, Amanda R.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Plant Sciences, University of California–Davis, Davis, CA.
    Li, Zhen
    Van de Peer, Yves
    Ingvarsson, Pär K.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Department of Plant Biology, Uppsala Biocenter, Swedish University of Agr icultural Sciences, Uppsala, Sweden.
    Contrasting Rates of Molecular Evolution and Patterns of Selection among Gymnosperms and Flowering Plants2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 6, p. 1363-1377Article in journal (Refereed)
    Abstract [en]

    The majority of variation in rates of molecular evolution among seed plants remains both unexplored and unexplained. Although some attention has been given to flowering plants, reports of molecular evolutionary rates for their sister plant clade (gymnosperms) are scarce, and to our knowledge differences in molecular evolution among seed plant clades have never been tested in a phylogenetic framework. Angiosperms and gymnosperms differ in a number of features, of which contrasting reproductive biology, life spans, and population sizes are the most prominent. The highly conserved morphology of gymnosperms evidenced by similarity of extant species to fossil records and the high levels of macrosynteny at the genomic level have led scientists to believe that gymnosperms are slow-evolving plants, although some studies have offered contradictory results. Here, we used 31,968 nucleotide sites obtained from orthologous genes across a wide taxonomic sampling that includes representatives of most conifers, cycads, ginkgo, and many angiosperms with a sequenced genome. Our results suggest that angiosperms and gymnosperms differ considerably in their rates of molecular evolution per unit time, with gymnosperm rates being, on average, seven times lower than angiosperm species. Longer generation times and larger genome sizes are some of the factors explaining the slow rates of molecular evolution found in gymnosperms. In contrast to their slow rates of molecular evolution, gymnosperms possess higher substitution rate ratios than angiosperm taxa. Finally, our study suggests stronger and more efficient purifying and diversifying selection in gymnosperm than in angiosperm species, probably in relation to larger effective population sizes.

  • 152.
    De La Torre, Amanda R.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Univ British Columbia, Dept Forest & Conservat Sci, Ctr Forest Conservat Genet, Vancouver, BC V6T 1Z4, Canada.
    Roberts, David R.
    Aitken, Sally N.
    Genome-wide admixture and ecological niche modelling reveal the maintenance of species boundaries despite long history of interspecific gene flow2014In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 23, no 8, p. 2046-2059Article in journal (Refereed)
    Abstract [en]

    The maintenance of species boundaries despite interspecific gene flow has been a continuous source of interest in evolutionary biology. Many hybridizing species have porous genomes with regions impermeable to introgression, conferring reproductive barriers between species. We used ecological niche modelling to study the glacial and postglacial recolonization patterns between the widely hybridizing spruce species Picea glauca and P.engelmannii in western North America. Genome-wide estimates of admixture based on a panel of 311 candidate gene single nucleotide polymorphisms (SNP) from 290 genes were used to assess levels of admixture and introgression and to identify loci putatively involved in adaptive differences or reproductive barriers between species. Our palaeoclimatic modelling suggests that these two closely related species have a long history of hybridization and introgression, dating to at least 21000years ago, yet species integrity is maintained by a combination of strong environmental selection and reduced current interspecific gene flow. Twenty loci showed evidence of divergent selection, including six loci that were both F-st outliers and associated with climatic gradients, and fourteen loci that were either outliers or showed associations with climate. These included genes responsible for carbohydrate metabolism, signal transduction and transcription factors.

  • 153.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Substrate specificity and inhibitor sensitivity of plant UDP-sugar producing pyrophosphorylasesManuscript (preprint) (Other academic)
  • 154.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Meng, Meng
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Gornicka, Agnieszka
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kleczkowski, Leszek A
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase2012In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, p. 39-45Article in journal (Refereed)
    Abstract [en]

    UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

  • 155.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies2017In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 6, p. 1093-1107Article in journal (Other academic)
    Abstract [en]

    UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

  • 156.
    Decker, Daniel
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Öberg, Christopher
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kleczkowski, Leszek A.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases2018In: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 13, no 8, article id e1507406Article in journal (Refereed)
    Abstract [en]

    UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

  • 157. Deem, A
    et al.
    Keszthelyi, Andrea
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Blackgrove, T
    Vayl, A
    Coffey, B
    Mathur, R
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Malkova, A
    Department of Biology, School of Science, IUPUI, Indianapolis, Indiana, United States of America.
    Break-induced replication is highly inaccurate2011In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885Article in journal (Refereed)
    Abstract [en]

    DNA must be synthesized for purposes of genome duplication and DNA repair. While the former is a highly accurate process, short-patch synthesis associated with repair of DNA damage is often error-prone. Break-induced replication (BIR) is a unique cellular process that mimics normal DNA replication in its processivity, rate, and capacity to duplicate hundreds of kilobases, but is initiated at double-strand breaks (DSBs) rather than at replication origins. Here we employed a series of frameshift reporters to measure mutagenesis associated with BIR in Saccharomyces cerevisiae. We demonstrate that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2,800-fold higher than during normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. We established that polymerase proofreading and mismatch repair correct BIR errors. Also, dNTP levels were elevated during BIR, and this contributed to BIR-related mutagenesis. We propose that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR, with Pol δ generating many of the mutagenic errors. We further postulate that activation of BIR in eukaryotic cells may significantly contribute to accumulation of mutations that fuel cancer and evolution.

  • 158. Dejonghe, Wim
    et al.
    Kuenen, Sabine
    Mylle, Evelien
    Vasileva, Mina
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Viotti, Corrado
    Swerts, Jef
    Fendrych, Matyas
    Ortiz-Morea, Fausto Andres
    Mishev, Kiril
    Delang, Simon
    Scholl, Stefan
    Zarza, Xavier
    Heilmann, Mareike
    Kourelis, Jiorgos
    Kasprowicz, Jaroslaw
    Nguyen, Le Son Long
    Drozdzecki, Andrzej
    Van Houtte, Isabelle
    Szatmari, Anna-Maria
    Majda, Mateusz
    Baisa, Gary
    Bednarek, Sebastian York
    Robert, Stephanie
    Audenaert, Dominique
    Testerink, Christa
    Munnik, Teun
    Van Damme, Daniel
    Heilmann, Ingo
    Schumacher, Karin
    Winne, Johan
    Friml, Jiri
    Verstreken, Patrik
    Russinova, Eugenia
    Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 11710Article in journal (Refereed)
    Abstract [en]

    ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

  • 159.
    Del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bernardo, Lisandro M D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Skärfstad, Eleonore
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmfeldt, Linda
    Togneri, Peter
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 14, p. 5853-5865Article in journal (Refereed)
    Abstract [en]

    The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.

  • 160.
    del Peso-Santos, Teresa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Inter-sigmulon communication through topological promoter coupling2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 20, p. 9638-9649Article in journal (Refereed)
    Abstract [en]

    Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative sigma-factors. This is the case for the non-overlapping sigma(70)- and sigma(54)-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl) phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the sigma(54)-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the sigma(70)-promoter. The non-overlapping 'back-to-back' configuration of a powerful sigma(54)-promoter and weak sigma(70)-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the sigma(70)-promoter subservient to signals that elicit sigma(54)-dependent transcription without it possessing a cognate binding site for the sigma(54)-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.

  • 161. Dellino, Gaetano I
    et al.
    Schwartz, Yuri B
    University of Geneva, Switzerlandc.
    Farkas, Gabriella
    McCabe, Donna
    Elgin, Sarah C R
    Pirrotta, Vincenzo
    Polycomb silencing blocks transcription initiation.2004In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 13, no 6, p. 887-93Article in journal (Refereed)
    Abstract [en]

    Polycomb (PcG) complexes maintain the silent state of target genes. The mechanism of silencing is not known but has been inferred to involve chromatin packaging to block the access of transcription factors. We have studied the effect of PcG silencing on the hsp26 heat shock promoter. While silencing does decrease the accessibility of some restriction enzyme sites to some extent, it does not prevent the binding of TBP, RNA polymerase, or the heat shock factor to the hsp26 promoter, as shown by chromatin immunoprecipitation. However, we find that in the repressed state, the RNA polymerase cannot initiate transcription. We conclude that, rather than altering chromatin structure to block accessibility, PcG silencing in this construct targets directly the activity of the transcriptional machinery at the promoter.

  • 162.
    Demakov, Sergei
    et al.
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Gortchakov, Andrei
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Schwartz, Yuri B
    Institute of Cytology & Genetics, Russian Academy of Sciences, Russia.
    Semeshin, Valery
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Campuzano, Sonsoles
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Modolell, Juan
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Zhimulev, Igor
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes.
    Molecular and genetic organization of Drosophila melanogaster polytene chromosomes: evidence for two types of interband regions2004In: Genetica, ISSN 0016-6707, E-ISSN 1573-6857, Vol. 122, no 3, p. 311-324Article in journal (Refereed)
    Abstract [en]

    The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P[1ArB] transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.

  • 163.
    den Hollander, Jürgen
    et al.
    III. Medical Department, Technische Universität München, Munich, Germany.
    Rimpi, Sara
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Doherty, Joanne R
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    Rudelius, Martina
    Department of Pathology, Technische Universität München, Munich, Germany.
    Buck, Andreas
    Department of Nuclear Medicine, Technische Universität München, Munich, Germany.
    Kremer, Marcus
    Department of Pathology, Technische Universität München, Munich, Germany.
    Graf, Nikolas
    III. Medical Department, Technische Universität München, Munich, Germany.
    Scheerer, Markus
    III. Medical Department, Technische Universität München, Munich, Germany.
    Hall, Mark
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    von Bubnoff, Nikolas
    III. Medical Department, Technische Universität München, Munich, Germany.
    Duyster, Justus
    III. Medical Department, Technische Universität München, Munich, Germany.
    Peschel, Christian
    III. Medical Department, Technische Universität München, Munich, Germany.
    Cleveland, John L
    Department of Cancer Biology, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, USA.
    Nilsson, Jonas A
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Keller, Ulrich
    III. Medical Department, Technische Universität München, Munich, Germany.
    Aurora kinases A and B are Myc targets essential for maintenance of the malignant stateManuscript (preprint) (Other academic)
  • 164. Desmarais, SN
    et al.
    Tropini, C
    Miguel, A
    Cava, Felipe
    MIMS / Umeå University.
    Monds, RD
    de Pedro, MA
    Huang, KC
    High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography.2015In: J Biol Chem., Vol. 25, no 290(52), p. 31090-31100Article in journal (Refereed)
  • 165.
    Devadas, Arun
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Characterization of fungual strains for bioethanol production and sugar utilization2012Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Bioethanol production from cellulose based sources is currently in focus in several research projects and the need of the hour is a versatile fermenting organism that can utilize both 5C and 6C sugars effectively. Several naturally occurring fungi have the property of fermenting both types of sugars more efficiently than the traditionally used Saccharomyces cerevisiae which only can ferment hexose sugars. In this study, six different fungi were grown on a media with sugar concentrations similar to the spent sulphite liquor (SSL) from the paper pulp industry. Known fungi such as S. cerevisiae and T. versicolor as well as 4 unidentified wood rot species were grown in sealed bottles with media containing mixture of 6C and 5C. Comparison was made between ethanol fermentation, sugar consumption and enzyme activities (ALDH and PDC). The fermentation experiment was run for 21 days and ethanol concentrations up to 18g/L were achieved. We conclude that two of the fungi produce sufficient amount of ethanol and could be used in large-scale fermentation processes.

  • 166.
    Dimotakis, Charilaos
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Understanding of the role of CAD4, CAD5 and CAD6 gene redundancy during the lignification of Arabidopsis xylem.2015Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    Abstract

     

    Lignin is a cell wall polyphenolic polymer constituting the second most abundant bio-polymer on earth. This polymer is mostly accumulated in wood or xylem where it covers the other cell wall polysaccharides. Thus the removal of lignin allows accessing the polysaccharidic cell wall polymers which can then be converted into bio-fuels, textile or paper. Understanding lignin biosynthesis is therefore important to improve the industrial processing of the woody biomass. Lignin derives from the oxidative polymerization of different types of monomers called monolignols including 4-hydroxyphenylpropene alcohols as the most common monomers. Monolignols derive from the amino acid phenylalanine which is converted by a biosynthetic pathway known as the phenylpropanoid pathway. The last step of the multiple enzymatic process use the enzyme cinnamyl alcohol dehydrogenase (CAD) to convert 4-hydroxyphenylpropene aldehydes into 4-hydroxyphenylpropene alcohols. CAD is encoded by a small multigenic family in Arabidopsis thaliana comprising 17 genes.

    The aim of this master thesis is to understand the role of cinnamyl alcohol dehydrogenase gene redundancy during xylem lignification. CAD4, CAD5 and CAD6 have been associated in previous studies with xylem lignification and the main aim is to decipher if these genes are redundant or if they exhibit specificity in their expression (level, time and/or localization) and/or protein activity and structure. To do so, a genetic analysis of the single and double T-DNA insertional loss-of-function mutants in each of these genes were studied to compare their morphological characteristics, their biochemical structure (for both lignin quantification and composition) as well as their gene expression levels.

    Although minor changes in the lignin quantity and composition were observed for all of the single mutants, double mutants exhibited significant reductions and changes. Gene expression analysis moreover showed that the loss-of-function in any one of the three CADs caused a reduction ranging from 48% to 95% of the expression of the other CADs independently of the gene mutated. CAD4 and CAD5 both catalyzed the reduction of classical 4-hydroxyphenylpropene aldehydes into their corresponding alcohols: CAD5 catalytic activity is more specific to doubly methoxylated 4-hydroxyphenylpropene aldehydes than CAD4. In contrast, CAD6 did not affect the classical monolignols incorporation into lignin, but instead appeared to assist the function of CAD4 and CAD5. A clear synergetic effect of the double mutants suggested that a potential interaction could occur between these CAD proteins. Overall, our analysis showed that these three CAD genes were not redundant, but instead exhibited distinct function during xylem lignin biosynthesis.

  • 167.
    Dingeldein, Artur Peter Günther
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zhong, Xueyin
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy.
    Stoll, Raphael
    Ruhr University of Bochum, Biomolecular NMR Spectroscopy.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bax to the future: A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studiesIn: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279Article in journal (Refereed)
    Abstract [en]

    Mitochondria-mediated apoptosis (programmed cell death) is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. The members of this family can be divided into pro- and anti-apoptotic functioning proteins. One of the most important members for executing apoptosis is the cytosolic apoptotic Bax protein. It is crucial in facilitating onset of apoptosis by disrupting and forming pores within the mitochondrial outer membrane (MOM). This pore formation is commonly viewed as a point of no return, since it releases apotogenic factors such as cytochrome c into the cytosol who trigger irreversibly cell death by caspase activation and nuclear fragmentation. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli due to the strong similarities between both membrane-active segments. Here, we present a novel approach to express and purify full-length Bax with significantly increased yields compared to the commonly applied strategy with very bad yields. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we rendered both Bax termini inactive and prevented disruptive interactions during gene expression. By deploying an Intein-CBD-tag at the C-terminus we avoided even the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the purification procedure. Yields for pure, full-length Bax protein are increased by an order of magnitude, compared to commonly used Bax expression protocols.

  • 168.
    Dinh, Ngoc Phuoc
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Jonsson, Tobias
    Merck SeQuant AB, Box 7956, S-90719 Umeå, Sweden.
    Irgum, Knut
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Probing the interaction mode in hydrophilic interaction chromatography2011In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1218, no 35, p. 5880-5891Article in journal (Refereed)
    Abstract [en]

    This work aims at characterizing interactions between a select set of probes and 22 hydrophilic and polar commercial stationary phases, to develop an understanding of the relationship between the chemical properties of those phases and their interplay with the eluent and solutes in hydrophilic interaction chromatography. "Hydrophilic interaction" is a somewhat inexact term, and an attempt was therefore made to characterize the interactions involved in HILIC as hydrophilic, hydrophobic, electrostatic, hydrogen bonding, dipole-dipole, π-π interaction, and shape-selectivity. Each specific interaction was quantified from the separation factors of a pair of similar substances of which one had properties promoting the interaction mode being probed while the other did not. The effects of particle size and pore size of the phases on retention and selectivity were also studied. The phases investigated covered a wide range of surface functional groups including zwitterionic (sulfobetaine and phosphocholine), neutral (amide and hydroxyl), cationic (amine), and anionic (sulfonic acid and silanol). Principal component analysis of the data showed that partitioning was a dominating mechanism for uncharged solutes in HILIC. However, correlations between functional groups and interactions were also observed, which confirms that the HILIC retention mechanism is partly contributed by adsorption mechanisms involving electrostatic interaction and multipoint hydrogen bonding. Phases with smaller pore diameters yielded longer retention of solutes, but did not significantly change the column selectivities. The particle diameter had no significant effect, neither on retention, nor on the selectivities. An increased water content in the eluent reduced the multipoint hydrogen bonding interactions, while an increased electrolyte concentration lowered the selectivities of the tested columns and made their interaction patterns more similar.

  • 169.
    Dinh, Ngoc Phuoc
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Merck SeQuant AB, Umeå.
    Jonsson, Tobias
    Merck SeQuant AB, Umeå.
    Irgum, Knut
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Water uptake on polar stationary phases under conditions for hydrophilic interaction chromatography and its relation to solute retention2013In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1320, p. 33-47Article in journal (Other academic)
    Abstract [en]

    In hydrophilic interaction chromatography, water is known to accumulate on the stationary phase to form a water enriched layer, which is believed to play an important role in the retention mechanism. To gain a better understanding retention mechanism in HILIC, we have determined the water uptake on twelve different HILIC stationary phases. Non-modified and monomerically functionalized silica phases followed a pattern of monolayer formation followed by multiple layer adsorption, while the water uptake on polymerically functionalized silica stationary phase showed the characteristics of formation and swelling of hydrogels. This difference in the nature of water accumulation was found to be related to different water uptake patterns when methanol and tetrahydrofuran were added to 80:20 % (v/v) acetonitrile/water by replacing 5 % of the acetonitrile as tertiary solvents, and also when ammonium acetate was added as buffering electrolyte. The relationship between water uptake and retention mechanism was investigated by looking at the correlation between retention factors of neutral analytes and phase ratios of HILIC columns, calculated either as surface area (adsorption) or volume of the water layer enriched from the acetonitrile/water eluent (partitioning). Regardless of the adsorption or partitioning mechanism, the interaction of neutral analytes and stationary phase could be mainly the hydrogen bonding between analytes and the accumulated water in the water enriched layer.

  • 170. Dixon, Christopher J
    et al.
    Schoenswetter, Peter
    Suda, Jan
    Wiedermann, Magdalena M
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences.
    Schneeweiss, Gerald M
    Reciprocal Pleistocene origin and postglacial range formation of an allopolyploid and its sympatric ancestors (Androsace adfinis group, Primulaceae)2009In: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 50, no 1, p. 74-83Article in journal (Refereed)
    Abstract [en]

    The biogeographic history of polyploids and their lower-ploid ancestors is an important feature to achieve a better understanding of polyploid evolution. This is exemplified here using the ecologically congruent members of the Androsace adfinis group (Primulaceae) endemic to the southwestern European Alps. Employing relative genome size, AFLP fingerprint and chloroplast sequence haplotype data, we show that Androsace brigantiaca is a recent (probably no more than 0.2 million years) allopolyploid derivative of the geographically close A adfinis and A puberula, which formed reciprocally in a comparatively restricted area in the southern Southwestern Alps. Bayesian admixture analysis-also of artificial additive AFLP profiles-shows that the nuclear genome of A. brigantiaca is significantly biased towards the puberula-genome irrespective of maternal parentage. Nevertheless, there is no evidence for genetic interaction (hybridization, introgression) of A brigantiaca with either of its ancestors, including the widely sympatric A. puberula. Sympatry might be facilitated by ecological displacement on a local scale or might be a transitory phase on the way to competitive replacement via, for instance, polyploid superiority.

  • 171.
    Do, Lan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Geladi, Paul
    Haglund, Peter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Multivariate data analysis to characterize gas chromatography columns for dioxin analysis2014In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1347, p. 137-145Article in journal (Refereed)
    Abstract [en]

    Principal component analysis (PCA) was applied for evaluating the selectivity of 22 GC columns for which complete retention data were available for the 136 tetra- to octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). Because the hepta- and octa-homologues are easy to separate the PCA was focused on the 128 tetra- to hexa-CDD/Fs. The analysis showed that 21 of the 22 GC columns could be subdivided into four groups with different selectivity. Group I consists of columns with non-polar thermally stable phases (Restek 5Sil MS and Dioxin 2, SGE BPX-DXN, Supelco Equity-5, and Agilent DB-1, DB-5, DB-5ms, VF-5ms, VF-Xms and DB-XLB). Group II includes ionic liquid columns (Supelco SLB-IL61, SLB-IL111 and SLB-IL76) with very high polarity. Group III includes columns with high-percentage phenyl and cyanopropyl phases (Agilent DB-17 and DB-225, Quadrex CPS-1, Supelco SP-2331, and Agilent CP-Sil 88), and Group IV columns with shape selectivity (Dionex SB-Smectic and Restek LC-50, Supelco beta DEXcst, Agilent VF-Xms and DB-XLB). Thus, two columns appeared in both Group I and IV (Agilent VF-Xms and DB-XLB). The selectivity of the other column, Agilent DB-210, differs from those of these four groups. Partial least squares (PLS) regression was used to correlate the retention times of the tetra- to hexa-CDD/Fs on the 22 stationary phases with a set of physicochemical and structural descriptors to identify parameters that significantly influence the solute-stationary phase interactions. The most influential physicochemical parameters for the interaction were associated with molecular size (as reflects in the total energy, electron energy, core-core repulsion and standard entropy), solubility (aqueous solubility and n-octanol/water partition coefficient), charge distribution (molecular polarizability and dipolar moment), and reactivity (relative Gibbs free energy); and the most influential structural descriptors were related to these parameters, in particular, size and dipolar moment. Finally, the PCA and PLS analyses were complemented with linear regression analysis to identify the most orthogonal column combinations, which could be used in comprehensive two-dimensional gas chromatography (GC x GC) to enhance PCDD/F separation and congener profiling. (C) 2014 Elsevier B.V. All rights reserved.

  • 172.
    Do, Lan
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Liljelind, Per
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Zhang, Jin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haglund, Peter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Comprehensive profiling of 136 tetra- to octa-polychlorinated dibenzo-p-dioxins and dibenzofurans using ionic liquid columns and column combinations2013In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1311, p. 157-169Article in journal (Refereed)
    Abstract [en]

    The orders of elution of all 136 tetra- to octa-chlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) were determined on six gas chromatography (GC) columns. Three of these columns had ionic liquid stationary phases (SLB-IL111, SLB-IL76 and SLB-IL61; Supelco), one a liquid crystal phase (LC-50; Restek), one a chiral phase (beta DEXcst; Restek) and one a low bleed non-polar phase (DB-XLB; J&W/Agilent). According to our results, the high polarity and multiple solvation interactions of the ionic liquid stationary phases offered superior resolving power to that achieved with previously evaluated columns. The SLB-IL61 and SLB-IL111 columns resolved or partially separated 106 and 100 congeners, respectively, of the 136 PCDD/Fs. The SLB-IL61 also resolved 15 and partially separated one of the seventeen 2,3,7,8-substituted PCDD/Fs. Additional congeners can be separated by complementary analyses using additional columns in a dual- or triple-column approach. For example, using a combination of the SLB-IL61 and SLB-IL111 columns all but 8 congeners would be separated, including all 2,3,7,8-substituted PCDD/Fs. Two more congeners would be separated using a combination of SLB-IL76 and a liquid crystal (SB-Smectic) column, but in this case the 2,3,7,8-TeCDF would not be resolved. Three-column combinations would give even better separation: the DB-17/Smectic/SLB-IL76 and DB-225/Smectic/SLB-IL111 combinations would separate all but 1 of the 136 PCDD/F congeners. Unfortunately, the smectic column is no longer in production. If only commercially available columns are considered, combinations of SLB-IL61 and SLB-IL111 with DB-XLB, LC-50, or DB-225 offer the best performance, with 4, 4, and 3 unresolved congeners, respectively. Moreover, in each of these cases, one of the congeners in each unresolved pair is resolved on at least one of the other columns and so a reasonable estimate of the unresolved congeners' concentrations can be obtained by subtraction. The profiling of all 136 PCDD/Fs is thus greatly facilitated by using ionic liquid columns or combinations including such columns. However, there is room for improvement in the technical performance of the evaluated ionic liquid columns: their long-term retention time stability was poor and some highly chlorinated and sterically hindered congeners underwent dehalogenation during separation.

    (C) 2013 Elsevier B.V. All rights reserved.

  • 173.
    Dobrenel, Thomas
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, Versailles, France; Université Paris-Sud–Université Paris-Saclay, Orsay, France.
    Mancera-Martinez, Eder
    Forzani, Celine
    Azzopardi, Marianne
    Davanture, Marlene
    Moreau, Manon
    Schepetilnikov, Mikhail
    Chicher, Johana
    Langella, Olivier
    Zivy, Michel
    Robaglia, Christophe
    Ryabova, Lyubov A.
    Hanson, Johannes
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Meyer, Christian
    The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S62016In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 7, article id 1611Article in journal (Refereed)
    Abstract [en]

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  • 174.
    Domar, Ulla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Human intestinal alkaline phosphatase: tissue expression and serum levels1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies.

    Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs.

    IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine.

    Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed.

    Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance.

    Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated.

    Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed.

  • 175. Domingo-Almenara, Xavier
    et al.
    Montenegro-Burke, J. Rafael
    Ivanisevic, Julijana
    Thomas, Aurelien
    Sidibé, Jonathan
    Teav, Tony
    Guijas, Carlos
    Aisporna, Aries E.
    Rinehart, Duane
    Hoang, Linh
    Nordström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gómez-Romero, Maria
    Whiley, Luke
    Lewis, Matthew R.
    Nicholson, Jeremy K.
    Benton, H. Paul
    Siuzdak, Gary
    CMS-MRM and METLIN-MRM: a cloud library and public resource for targeted analysis of small molecules2018In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 15, no 9, p. 681-+Article in journal (Refereed)
    Abstract [en]

    We report XCMS-MRM and METLIN-MRM (http://xcmsonline-mrm.scripps.edu/ and http://metlin.scripps.edu/), a cloud-based data-analysis platform and a public multiple-reaction monitoring (MRM) transition repository for small-molecule quantitative tandem mass spectrometry. This platform provides MRM transitions for more than 15,500 molecules and facilitates data sharing across different instruments and laboratories.

  • 176. Dongre, Mitesh
    et al.
    Khatri, Neelam
    Dureja, Chetna
    Raychaudhuri, Saumya
    Alanine-scanning mutagenesis of selected residues in the N-terminal region alters the functionality of LuxO: lessons from a natural variant LuxOPL91.2011In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 60, no Pt 6, p. 856-60Article in journal (Refereed)
  • 177. Dongre, Mitesh
    et al.
    Nyunt Wai, Sun
    Chapter 273 - The Protease of Vibrio cholerae (PrtV) A2 - Rawlings, Neil D.2013In: Handbook of Proteolytic Enzymes, Academic Press , 2013, p. 1219-1225Chapter in book (Other academic)
    Abstract [en]

    The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 850 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 273 is The Protease of Vibrio cholerae (PrtV). Keywords Auto-proteolysis, Caenorhabditis elegans, cytokine induction, fibrinogen, fibronectin, HapR. Vibrio cholerae cytolysin (VCC), M6-peptidase family, plasminogen, polycystic kidney disease (PKD) domains, Quorum Sensing (QS), zinc-dependent metalloproteases.

  • 178. Dongre, Mitesh
    et al.
    Singh, Naorem Santa
    Dureja, Chetna
    Peddada, Nagesh
    Solanki, Ashish K
    Ashish, Ganguly
    Raychaudhuri, Saumya
    Evidence on how a conserved glycine in the hinge region of HapR regulates its DNA binding ability: lessons from a natural variant.2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 17, p. 15043-9Article in journal (Refereed)
    Abstract [en]

    HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapR(V2)) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapR(V2) to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapR(V2) (HapR(V2G)), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapR(V2) and HapR(V2G) proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a "Y" shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

  • 179. Dongre, Mitesh
    et al.
    Tripathi, Ranjana
    Jain, Vibhu
    Raychaudhuri, Saumya
    Functional independence of a variant LuxOPL91 from a non-O1 non-O139 Vibrio cholerae over the activity of CsrA and Fis.2008In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 57, no Pt 8, p. 1041-5Article in journal (Refereed)
  • 180.
    Dorafshan, Eshagh
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kahn, Tatyana G.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schwartz, Yuri B.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hierarchical recruitment of Polycomb complexes revisited2017In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 8, no 5, p. 496-505Article in journal (Refereed)
    Abstract [en]

    Polycomb Group (PcG) proteins epigenetically repress key developmental genes and thereby control alternative cell fates. PcG proteins act as complexes that can modify histones and these histone modifications play a role in transmitting the memory of the repressed state as cells divide. Here we consider mainstream models that link histone modifications to hierarchical recruitment of PcG complexes and compare them to results of a direct test of interdependence between PcG complexes for recruitment to Drosophila genes. The direct test indicates that PcG complexes do not rely on histone modifications to recognize their target genes but use them to stabilize the interactions within large chromatin domains. It also shows that multiple strategies are used to coordinate the targeting of PcG complexes to different genes, which may make the repression of these genes more or less robust.

  • 181. Drotz, Marcus K.
    et al.
    Savolainen, Eino
    Saura, Anssi
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Stahls, Gunilla
    The genetic population structure of lotic and lentic mayflies of the Baetis vernus group (Ephemeroptera: Baetidae)2012In: Canadian Entomologist, ISSN 0008-347X, E-ISSN 1918-3240, Vol. 144, no 5, p. 679-690Article in journal (Refereed)
    Abstract [en]

    Nymphs of lotic mayflies live in environments that are expected to give rise to different degrees of population structuring. Here we investigate two taxa adapted to different lifestyles. Baetis macani Kimmins (Ephemeroptera: Baetidae) lives in flowing water; brooks that may periodically dry out in the summer or freeze to the bottom in winter. Baetis jaervii Savolainen is mostly found in sedge belts along the shores of lakes. Most insects living in flowing water show low levels of among-population genetic differentiation within and among catchments. Levels of differentiation in the lotic species are therefore assumed to be lower than in lentic B. jaervii. Here we test this hypothesis. Mitochondrial DNA and allele frequencies of nuclear genes were used to detect population structure in specimens originating from an extensive area from northern Finland. The genetic differentiation among populations of the lotic B. macani is more than twice the corresponding value for the lentic B. jaervii (F-ST 0.33 versus 0.15, while the mean F-ST between species was 0.33 and significant). The result is congruent within the cytochrome c oxidase subunit I gene (COI) partial gene frequencies. We argue that the significant genetic population structure, which only was found in the lotic B. macani, is differentiated as a consequence to the unpredictable environment as contrasted to the stable environment in standing bodies of water.

  • 182. Du, Shuhui
    et al.
    Wang, Zhaoshan
    Ingvarsson, Pär K
    Umeå University, Faculty of Science and Technology, Department of Ecology and Environmental Sciences. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Wang, Dongsheng
    Wang, Junhui
    Wu, Zhiqiang
    Tembrock, Luke R.
    Zhang, Jianguo
    Multilocus analysis of nucleotide variation and speciation in three closely related Populus (Salicaceae) species2015In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 24, no 19, p. 4994-5005Article in journal (Refereed)
    Abstract [en]

    Historical tectonism and climate oscillations can isolate and contract the geographical distributions of many plant species, and they are even known to trigger species divergence and ultimately speciation. Here, we estimated the nucleotide variation and speciation in three closely related Populus species, Populus tremuloides, P.tremula and P.davidiana, distributed in North America and Eurasia. We analysed the sequence variation in six single-copy nuclear loci and three chloroplast (cpDNA) fragments in 497 individuals sampled from 33 populations of these three species across their geographic distributions. These three Populus species harboured relatively high levels of nucleotide diversity and showed high levels of nucleotide differentiation. Phylogenetic analysis revealed that P.tremuloides diverged earlier than the other two species. The cpDNA haplotype network result clearly illustrated the dispersal route from North America to eastern Asia and then into Europe. Molecular dating results confirmed that the divergence of these three species coincided with the sundering of the Bering land bridge in the late Miocene and a rapid uplift of the Qinghai-Tibetan Plateau around the Miocene/Pliocene boundary. Vicariance-driven successful allopatric speciation resulting from historical tectonism and climate oscillations most likely played roles inthe formation of the disjunct distributions and divergence of these three Populus species.

  • 183.
    Dwivedi, Gaurav Dutta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Cloning and Expression of Streptococcal Recombinant Protein G.2015Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    •  
  • 184.
    Dzhygyr, Ievgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Functional studies of Escherichia coli stringent response factor RelA2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    RelA is a ribosome associated multi-domain enzyme, which plays a crucial role in adaptation ofEscherichia coli to nutritional stress as such as amino acid deficiency. It detects the deficiency of aminoacids in the cell by monitoring whether a tRNA at the acceptor site (A-site) of the ribosome is chargedwith amino acid or not. When RelA detects uncharged, i.e. deacylated tRNA, it starts to producealarmone guanosine penta- or tetraphosphate, collectively referred to as (p)ppGpp. (p)ppGpp is aglobal metabolism regulator in bacteria. Increase in (p)ppGpp concentration alters crucial metabolicprocesses, such as DNA replication, gene expression, cell wall synthesis and translation. Thesechanges also include activation of different virulence factors and are proposed to drive formation of abacterial sub-population that is highly resilient to antibiotic treatment, the so-called persisters.

    For a long time the molecular mechanism of RelA’s activation by and interaction with the ribosomedeacylatedtRNA complex was unknown. Only recently several cryo-EM structures of RelA-ribosomecomplex have shed light on how C-terminal domains of RelA interact with ribosome-deacylated tRNAcomplex. Guided by these structures we investigated the role of RelA’s domains in this interaction byconstructing a set of RelA C-terminal truncates and subjecting these to biochemical and microbiologicalexperimentation. These experiments were complemented with mutations in ribosomal RNA atpositions that interact with RelA, namely A-site finger and sarcin-ricin loop.

    We have shown that only the full-length wild type RelA can be activated by ribosome-tRNA complex,whereas, the set of truncated proteins missing either one, two or three C-terminal domains do notrespond to the presence of uncharged tRNA in the A-site of the ribosome. However, these truncatedversions can still be activated by vacant 70S ribosome as well as pppGpp, suggesting that N-terminaldomain of RelA has an allosteric regulation site for (p)ppGpp and is able to interact with the ribosome.The mechanism of this interaction is yet to be elucidated.

    We have shown that A-site finger of the ribosome is required for RelA activation and recruitment tothe ribosome. Using EMSA assays, we have shown that RelA and deacylated tRNA do not form a stablecomplex off the ribosome. His432 located in TGS domain of RelA is crucial for recognition of deacylatedtRNA and a mutation of this histidine to glycine abolishes RelA activation by deacylated tRNA.Since (p)ppGpp plays an important role in bacterial survival and pathogenicity we have also testedseveral strategies for RelA inhibition by antibiotics, which target ribosomes and the interactionbetween RelA and ribosome-deacylated tRNA complex. We have shown that antibiotic thiostreptoninhibits (p)ppGpp synthesis by preventing RelA-tRNA interaction on the ribosome. (p)ppGppproduction is also inhibited by chloramphenicol and tetracycline.

  • 185.
    Dzhygyr, Ievgen
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Cut-back analysis reveals the role of RelA's individual domains in its activation by 'starved' ribosomal complexes and pppGpp alarmone nucleotideManuscript (preprint) (Other academic)
  • 186. Díaz-Salazar, Carlos
    et al.
    Calero, Patricia
    Espinosa-Portero, Rocío
    Jiménez-Fernández, Alicia
    Wirebrand, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Velasco-Domínguez, María G.
    López-Sánchez, Aroa
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Govantes, Fernando
    The stringent response promotes biofilm dispersal in Pseudomonas putida2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 18055Article in journal (Refereed)
    Abstract [en]

    Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response – (p)ppGpp sythetases [RelA and SpoT] and/or DksA – were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

  • 187. Edman, Maria
    et al.
    Berg, Stefan
    Storm, Patrik
    Wikström, Malin
    Vikström, Susanne
    Öhman, Anders
    Wieslander, Ake
    Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae.2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 10Article in journal (Refereed)
    Abstract [en]

    In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.

  • 188. Edqvist, Petra
    et al.
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Steggo, Peter
    Forsberg, Åke
    Francis, Matthew
    Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificityManuscript (Other academic)
  • 189.
    Edqvist, Petra J
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

    The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

    Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

    Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

  • 190.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew S
    Examination of LcrH type III secretion chaperone function during Yersinia-eukaryotic cell contactManuscript (Other academic)
  • 191.
    Edqvist, Petra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lavander, Moa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Sundberg, Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lloyd, Scott
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System2003In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 185, no 7, p. 2259-2266Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

  • 192.
    Eerola, Iiro
    et al.
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Salminen, Heli
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Lammi, Pirkko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    von der Mark, Klaus
    Institute of Experimental Medicine, Friedrich Alexander University of Erlangen-Nuremberg, Erlangen, Germany.
    Vuorio, Eero
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Säämänen, Anna-Marja
    Department of Medical Biochemistry and Molecular Biology, University of Turku, Turku, Finland.
    Type X collagen, a natural component of mouse articular cartilage: association with growth, aging, and osteoarthritis.1998In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 41, no 7, p. 1287-1295, article id 9663487Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: To perform a systematic study on the production and deposition of type X collagen in developing, aging, and osteoarthritic (OA) mouse articular cartilage.

    METHODS: Immunohistochemistry was employed to define the distribution of type X collagen and Northern analyses to determine the messenger RNA levels as an indicator of the synthetic activity of the protein.

    RESULTS: Type X collagen was observed in the epiphyseal and articular cartilage of mouse knee joints throughout development and growth. Type X collagen deposition in the transitional zone of articular cartilage became evident toward cessation of growth, at the age of 2-3 months. The most intense staining for type X collagen was limited to the tidemark, the border between uncalcified and calcified cartilage. Northern analysis confirmed that the type X collagen gene is also transcribed by articular cartilage chondrocytes. Intense immunostaining was observed in the areas of OA lesions, specifically, at sites of osteophyte formation and surface fibrillation. Type X collagen deposition was also seen in degenerating menisci.

    CONCLUSION: This study demonstrates that type X collagen is a natural component of mouse articular cartilage throughout development, growth, and aging. This finding and the deposition of type X collagen at sites of OA lesions suggest that type X collagen may have a role in providing structural support for articular cartilage.

  • 193.
    Einarsdottir, Elisabet
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Söderström, Ingegerd
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Löfgren-Burström, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Unit for Genome Research, Umeå University.
    Haraldsson, Susann
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Unit for Genome Research, Umeå University.
    Nilsson-Ardnor, Sofie
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Unit for Genome Research, Umeå University.
    Penha-Goncalves, Carlos
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Gulbenkian Institute for Science, Oeiras, Portugal.
    Lind, Lisbet
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics. Unit for Genome Research, Umeå University.
    Holmgren, Gösta
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Holmberg, Monica
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Asplund, Kjell
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Holmberg, Dan
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Unit for Genome Research.
    The CTLA4 region as a general autoimmunity factor: an extended pedigree provides evidence for synergy with the HLA locus in the etiology of type 1 diabetes mellitus, Hashimoto's thyroiditis and Graves' disease2003In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 11, no 1, p. 81-84Article in journal (Refereed)
    Abstract [en]

    We have identified a large family in the northern part of Sweden with multiple cases of autoimmune diseases, namely type 1 diabetes (T1D), Graves' disease (GD) and Hashimoto's thyroiditis (HT). The family members affected by any of these diseases share a region of 2.4 Mb that comprises among others the CTLA4 gene. We determined that all affected members of the family shared the HLA susceptibility haplotype (DR4-DQA1*0301-DQB1*0302). Analysis of genetic interaction conditioning for HLA haplotype provided strong evidence that the critical region which includes the CTLA4 gene acts together with the HLA locus on the etiology of disease (lodscore 4.20 (theta=0.0). The study of this family allowed us to: (1) reinforce a number of reports on linkage and association of the CTLA4 region to T1D and AITD; (2) demonstrate that a single haplotypic variant in this region constitutes an etiological factor to disease susceptibility in T1D, GD and HT; (3) reveal a strong genetic interaction of the CTLA4 and HLA loci in the genetic architecture of autoimmune disease; (4) emphasise the value of large pedigrees drawn from isolated populations as tools to single out the effect of individual loci in the etiology of complex diseases.

  • 194.
    Ekestubbe, Sofie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Timing and targeting of Type III secretion translocation of virulence effectors in Yersinia2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.

  • 195.
    Ekström, Fredrik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    X-ray characterization of PaPheOH, a bacterial phenylalanine hydroxylase2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Many human diseases are associated with the malfunction of enzymes in the aromatic amino acid hydroxylase family, e.g. phenylketonuria (PKU), hyperphenylalaninemia (HPA), schizophrenia and Parkinson's disease. The family of aromatic aminoacid hydroxylases comprises the enzymes phenylalanine hydroxylase (PheOH), tyrosine hydroxylase (TyrOH) and tryptophane hydroxylase (TrpOH). These enzymes require the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and atomic oxygen. In eukaryotes, the aromatic amino acid hydroxylases share the same organization with a N-terminal regulatory domain, a central catalytic domain and a C-terminal tetramerization domain. Aromatic amino acid hydroxylases that correspond to the core catalytic domain of the eukaryotic enzymes are found in bacteria. The main focus of this thesis is the structural characterization of a phenylalanine hydroxylase from the bacterium Pseudomonas aeruginosa (PaPheOH).

    To initiate the structural characterization, the active site environment was investigated with X-ray absorption spectroscopy (XAS). The experimental data support a model where the active site iron is coordinated by four oxygen atoms and two nitrogen atoms. We suggest that two water molecules, His121, His126 and Glu166 coordinates the active site iron. In this model, Glu166 provides two of the oxygen atoms in a bidentate binding geometry. EXAFS and XANES studies indicate that structural rearrangements are induced in the second and third coordination shells in samples of PaPheOH with BH4 and/or L-Phe.

    The 1.6 Å X-ray structure of PaPheOH shows a catalytic core that is composed of helices and strands in a bowl-like arrangement. The iron is octahedrally coordinated, by two water molecules and the evolutionary conserved His121, His126 and Glu166 that coordinates the iron with bidentate geometry. The pterin binding loop of PaPheOH (residue 81-86) adopts a conformation that is displaced by 5-6 Å from the expected pterin binding site. Consistent with the unfavourable position of the pterin binding loop is the observation that PaPheOH has a low specific activity compared to the enzymes from human and Chromobacterium violaceum.

    The second part of this thesis focus on the crystallization and structure determination of the actin binding domain of a-actinin (ABD). a-Actinin is located in the Z-disc of skeletal muscle were it crosslinks actin filaments to the filamentous protein titin. The ABD domain of a-actinin crystallizes in space group P21 with four molecules in the asymmetric unit. The structure of the ABD domain has been solved to a d-spacing of 2.0 Å. The two CH-domains of ABD is composed of 5 a-helices each. The a-helices fold into a closed compact conformation with extensive intramolecular contacts between the two domains.

  • 196.
    Elo, Mika
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Kaarniranta, Kai
    Department of Opthalmology, Kuopio University Hospital, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Hsp90 inhibitor geldanamycin increases hsp70 mRNA stabilisation but fails to activate HSF1 in cells exposed to hydrostatic pressure.2005In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1743, no 1-2, p. 115-119, article id 15777846Article in journal (Refereed)
    Abstract [en]

    High hydrostatic pressure (HP) increases Hsp70 protein and mRNA levels by increasing the mRNA half-life without activation of HSF1 transcription factor. We investigated whether this change in gene expression requires Hsp90, previously shown to regulate hsp70 genes via HSF1. In HeLa cells, both HP and Hsp90 inhibitor geldanamycin (GA) up-regulated Hsp70 expression through mRNA stabilisation. GA, unlike HP, increased HSF1 activation. However, when exposures were used together a marked Hsp70 response was observed with mRNA stabilisation without coincidence of HSF1 activation. Our data suggests that Hsp90 is involved in hsp70 mRNA stabilisation and the HSF1 activation can be suppressed by high HP.

  • 197.
    Elo, Mika
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Karjalainen, Hannu
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Sironen, Reijo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Valmu, Leena
    Institute of Biotechnololgy, Biocenter Viikki, University of Helsinki, Helsinki, Finland.
    Redpath, Nicholas
    Celltech R & D, Slough, United Kingdom.
    Browne, Gareth
    Division of Molecular Physiology, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
    Kalkkinen, Nisse
    Institute of Biotechnololgy, Biocenter Viikki, University of Helsinki, Helsinki, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    High hydrostatic pressure inhibits the biosynthesis of eukaryotic elongation factor-2.2005In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 94, no 3, p. 497-507, article id 15534876Article in journal (Refereed)
    Abstract [en]

    High continuous hydrostatic pressure is known to inhibit the total cellular protein synthesis. In this study, our goal was to identify pressure-regulated proteins by using two dimensional gel electrophoresis and mass spectrometry. This analysis showed that under 30 MPa continuous hydrostatic pressure the biosynthesis of eukaryotic elongation factor-2 (eEF-2) was inhibited both in HeLa carcinoma and T/C28a4 chondrocytic cell lines. Western blot analysis of HeLa cells revealed that the cellular protein level of eEF-2 decreased by 40%-50% within 12 h of the pressure treatment. However, the steady-state mRNA level of eEF-2 was not affected by the pressure. Cycloheximide addition after 4 h-pressure treatment suggested that the half-life of eEF-2 protein was shorter in pressurized cells. eEF-2 is responsible for the translocation of ribosome along the specific mRNA during translation, and its phosphorylation prevents the ribosomal translocation. Therefore, increased phosphorylation of eEF-2 was considered as one mechanism that could explain the reduced level of protein synthesis in pressurized HeLa cell cultures. However, Western blot analysis with an antibody recognizing the Thr56-phosphorylated form of eEF-2 showed that phosphorylation of eEF-2 was not elevated in pressurized samples. In conclusion, the inhibition of protein synthesis under high pressure occurs independent of the phosphorylation of eEF-2. However, this inhibition may result from the decrease of cellular eEF-2 protein.

  • 198.
    Elo, Mika
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Sironen, Reijo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Kaarniranta, Kai
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Auriola, Seppo
    Department of Pharmaceutical Chemistry, University of Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Differential regulation of stress proteins by high hydrostatic pressure, heat shock, and unbalanced calcium homeostasis in chondrocytic cells.2000In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 79, no 4, p. 610-619, article id 10996852Article in journal (Refereed)
    Abstract [en]

    High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.

  • 199.
    Elo, Mika
    et al.
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Sironen, Reijo
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Karjalainen, Hannu
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Kaarniranta, Kai
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Specific induction of heat shock protein 90beta by high hydrostatic pressure.2003In: Biorheology, ISSN 0006-355X, E-ISSN 1878-5034, Vol. 40, no 1-3, p. 141-146, article id 12454398Article in journal (Refereed)
    Abstract [en]

    In chondrocytes, a low-amplitude intermittent hydrostatic pressure induces production of extracellular matrix molecules, while high hydrostatic pressure inhibits it. High pressure increases cellular heat shock protein 70 level in a number of cell types on account of increased stabilisation of the heat shock protein 70 mRNA. In our experiments, only bovine primary chondrocytes, but not an immortalized chondrocytic cell line, could resist the induction of the stress response in the presence of continuous 30 MPa hydrostatic pressure. We have recently shown that protein synthesis is required for the stabilization. According to two-dimensional gel electrophoresis the synthesis of heat shock protein 90 was also increased in a chondrocytic cell line and in HeLa cells, and mass spectrometric analysis suggested that the induction was rather due to increase in heat shock protein 90beta than in heat shock protein 90alpha. The stress response was rather intense in HeLa cells, therefore, we investigated the effect of continuous 30 MPa hydrostatic pressure on the expression of the two heat shock protein 90 genes in HeLa cells using Northern and Western blot analyses. Heat shock protein 90beta mRNA level increased within 6 hours of exposure to 30 MPa hydrostatic pressure, while hsp90alpha level remained stable. At protein level there was a clear increase in the heat shock protein 90beta/heat shock protein 90alpha ratio, too. These results show a specific regulation of stress proteins in cells exposed to high hydrostatic pressure.

  • 200.
    Eneqvist, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Lundberg, Erik
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Nilsson, Lars
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    Abagyan, Ruben
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
    The transthyretin-related protein family2003In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 270, no 3, p. 518-532Article in journal (Refereed)
    Abstract [en]

    A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.

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