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  • 151.
    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.

  • 152.
    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.

  • 153.
    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.

  • 154. 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.

  • 155.
    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.

  • 156.
    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.

  • 157.
    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.

  • 158.
    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.

  • 159. 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)
  • 160. 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.

  • 161. 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.

  • 162. 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)
  • 163.
    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.

  • 164. 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.

  • 165. 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.

  • 166.
    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]

    •  
  • 167. 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.

  • 168. 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.

  • 169.
    Edqvist, Petra J
    Umeå University, Faculty of Medicine, Molecular Biology.
    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.

  • 170.
    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.

  • 171.
    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.

  • 172.
    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.

  • 173.
    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.

  • 174.
    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.

  • 175.
    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.

  • 176.
    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.

  • 177.
    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.

  • 178.
    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.

  • 179.
    Engdahl, Cecilia
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Selective inhibition of acetylcholinesterase 1 from disease-transmitting mosquitoes: design and development of new insecticides for vector control2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

  • 180.
    Engelken, Johannes
    et al.
    Department of Biology, University of Konstanz, Constance, Germany .
    Funk, Christiane
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Adamska, Iwona
    Department of Biology, University of Konstanz, Constance, Germany.
    The extended light-harvesting complex (LHC) protein superfamily: Classification and evolutionary dynamics2012In: Functinal genomics and evolution of photosynthetic systems / [ed] Robert Burnap, Wim Vermaas, Dordrecht, Netherlands: Springer Netherlands, 2012, p. 265-284Chapter in book (Refereed)
    Abstract [en]

    The evolution of algae and land plants and their photosynthetic machineries is closely connected to the development of the extended light-harvesting complex (LHC) protein superfamily. Therefore, it is not surprising that the molecular organization, function and origin of the LHC proteins have been a central topic in plant biology and photosynthesis research during the last few years. The extended LHC protein superfamily in cyanobacteria and photosynthetic eukaryotes comprises different families, such as the LHC proteins and three groups of light stress-induced proteins, consisting of the LHC-like proteins, the red lineage CAB-like proteins and the photosystem II subunit S. This chapter provides a description of the different extended LHC superfamily members and shows their taxonomic distribution. Furthermore, an overview of scenarios suggested for the evolution of the extended LHC protein superfamily is provided and arised implications for light harvesting, stress responses and photoprotection are discussed.

  • 181.
    Engström, Patrik
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nguyen, Bidong D.
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Ingela
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bastidas, Robert J.
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Elofsson, Mikael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fields, Kenneth A.
    Valdivia, Raphael H.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity2013In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 195, no 18, p. 4221-4230Article in journal (Refereed)
    Abstract [en]

    Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.

  • 182.
    Enquist, Per-Anders
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Hägglund, Ulrik
    Creative Antibiotics, Tvistevägen 48, SE90719 Umeå, Sweden.
    Lindström, Pia
    Creative Antibiotics, Tvistevägen 48, SE90719 Umeå, Sweden.
    Norberg-Scherman, Henrik
    Creative Antibiotics, Tvistevägen 48, SE90719 Umeå, Sweden.
    Sundin, Charlotta
    Creative Antibiotics, Tvistevägen 48, SE90719 Umeå, Sweden.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Derivatives of 8-hydroxyquinoline-antibacterial agents that target intra- and extracellular Gram-negative pathogens2012In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 22, no 10, p. 3550-3553Article in journal (Refereed)
    Abstract [en]

    Small molecule screening identified 5-nitro-7-((4-phenylpiperazine-1-yl-)methyl)quinolin-8-ol INP1750 as a putative inhibitor of type III secretion (T3S) in the Gram-negative pathogen Yersinia pseudotuberculosis. In this study we report structure-activity relationships for inhibition of T3S and show that the most potent compounds target both the extracellular bacterium Y. pseudotuberculosis and the intracellular pathogen Chlamydia trachomatis in cell-based infection models.

  • 183.
    Erhagen, Björn
    et al.
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Öquist, Mats
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Sparrman, Tobias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Haei, Mahsa
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Ilstedt, Ulrik
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Hedenström, Mattias
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Schleucher, Jürgen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nilsson, Mats B
    Department of Forest Ecology & Management, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden.
    Temperature response of litter and soil organic matter decomposition is determined by chemical composition of organic material2013In: Global Change Biology, ISSN 1354-1013, E-ISSN 1365-2486, Vol. 19, no 12, p. 3858-3871Article in journal (Refereed)
    Abstract [en]

    The global soil carbon pool is approximately three times larger than the contemporary atmospheric pool, therefore even minor changes to its integrity may have major implications for atmospheric CO2 concentrations. While theory predicts that the chemical composition of organic matter should constitute a master control on the temperature response of its decomposition, this relationship has not yet been fully demonstrated. We used laboratory incubations of forest soil organic matter (SOM) and fresh litter material together with NMR spectroscopy to make this connection between organic chemical composition and temperature sensitivity of decomposition. Temperature response of decomposition in both fresh litter and SOM was directly related to the chemical composition of the constituent organic matter, explaining 90% and 70% of the variance in Q10 in litter and SOM respectively. The Q10 of litter decreased with increasing proportions of aromatic and O-aromatic compounds, and increased with increased contents of alkyl- and O-alkyl carbons. In contrast, in SOM, decomposition was affected only by carbonyl compounds. To reveal why a certain group of organic chemical compounds affected the temperature sensitivity of organic matter decomposition in litter and SOM, a more detailed characterisation of the (13) C aromatic region using Heteronuclear Single Quantum Coherence (HSQC) was conducted. The results revealed considerable differences in the aromatic region between litter and SOM. This suggests that the correlation between chemical composition of organic matter and the temperature response of decomposition differed between litter and SOM. The temperature response of soil decomposition processes can thus be described by the chemical composition of its constituent organic matter, this paves the way for improved ecosystem modelling of biosphere feedbacks under a changing climate.

  • 184. Eriksson, Hanna M
    et al.
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Zhang, Shuguang
    Wieslander, Åke
    High-yield expression and purification of a monotopic membrane glycosyltransferase2009In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 2, p. 143-148Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-dodecyl-beta-D-maltoside (DDM) (approximately 1x critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.

  • 185.
    Eriksson, Jonas
    et al.
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology (SCFAB), Stockholm.
    Gharizadeh, Baback
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology (SCFAB), Stockholm.
    Nordström, Tommy
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology (SCFAB), Stockholm.
    Nyrén, Pål
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology (SCFAB), Stockholm.
    Pyrosequencing trade mark technology at elevated temperature2004In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 1, p. 20-27Article in journal (Refereed)
    Abstract [en]

    To date, the Pyrosequencing trade mark technology has been performed at 28 degrees C due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37 degrees C. By increasing the temperature to 37 degrees C, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 M glycine betaine, and (iii) an increase of the temperature to 37 degrees C enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA em leader 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.

  • 186.
    Eriksson, Jonas
    et al.
    Department of Biotechnology, SCFAB (Stockholm Centre for Physics, Astronomy, and Biotechnology), KTH (Royal Institute of Technology), Stockholm.
    Gharizadeh, Baback
    Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, California.
    Nourizad, Nader
    Department of Biotechnology, SCFAB (Stockholm Centre for Physics, Astronomy, and Biotechnology), KTH (Royal Institute of Technology), Stockholm.
    Nyrén, Pål
    Department of Biotechnology, SCFAB (Stockholm Centre for Physics, Astronomy, and Biotechnology), KTH (Royal Institute of Technology), Stockholm.
    7-Deaza-2'-deoxyadenosine-5'-triphosphate as an alternative nucleotide for the pyrosequencing technology2004In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 23, no 10, p. 1583-1594Article in journal (Refereed)
    Abstract [en]

    A new adenosine nucleotide analog suitable for the Pyrosequencing method is presented. The new analog, 7-deaza-2'-deoxyadenosine-5'-triphosphate (c7dATP), has virtually the same low substrate specificity for luciferase as the currently used analog, 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) (dATPalphaS). The inhibitory effect dATPalphaS displays on the nucleotide degrading activity of apyrase was reduced significantly by substituting the c7dATP for the dATPalphaS. Both analogs show high stability after long time storage at + 8 degrees C. Furthermore, with the new nucleotide a read length of up to 100 bases was obtained for several templates from fungi, bacteria and viruses.

  • 187.
    Eriksson, Jonas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Biotechnology, Royal Institute of Technology, Stockholm.
    Karamohamed, S
    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts.
    Nyrén, P
    Department of Biotechnology, Royal Institute of Technology, Stockholm.
    Method for real-time detection of inorganic pyrophosphatase activity2001In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 293, no 1, p. 67-70Article in journal (Refereed)
    Abstract [en]

    A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.

  • 188.
    Eriksson, Jonas
    et al.
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Nordström, Tommy
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Nyrén, Pål
    Department of Biotechnology, AlbaNova University Center, SCFAB, Royal Institute of Technology, Stockholm.
    Method enabling firefly luciferase-based bioluminometric assays at elevated temperatures2003In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 314, no 1, p. 158-161Article in journal (Refereed)
  • 189.
    Eriksson, Kåre
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Occupational and Environmental Medicine.
    Östin, Anders
    National Institute for Working Life, Umeå.
    Levin, Jan-Olof
    National Institute for Working Life, Umeå.
    Quantification of melatonin in human saliva by liquid chromatography-tandem mass spectrometry using stable isotope dilution2003In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 794, no 1, p. 115-123Article in journal (Refereed)
    Abstract [en]

    A method for the determination of melatonin in human saliva has been developed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS). Saliva was collected in plastic tubes. 7-D-Melatonin was added as internal standard and the samples were cleaned and concentrated by solid-phase extraction. The limit of detection was 1.05 pg x ml(-1) and the limit of quantification was 3.0 pg x ml(-1). The accuracy of the method was +/-14% at 5.60 pg x ml(-1) and +/-9% at 19.6 pg x ml(-1). The precision was +/-13% at 6.18 pg x ml(-1) and +/-11% at 31.2 pg x ml(-1), respectively. Our HPLC-MS-MS method shows a high sensitivity and specificity for melatonin and more reliable results compared with a radioimmunoassay. The chromatographic method has been used to determine the circadian rhythm of melatonin among three nurses working the night shift and a patient suffering from an inability to fall asleep at night.

  • 190. Eriksson, Lennart
    et al.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Erik
    Bro, Rasmus
    Wold, Svante
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Orthogonal signal correction, wavelet analysis, and multivariate calibration of complicated process fluorescence data2000In: Analytica Chimica Acta, Vol. 420, no 2, p. 181-95Article in journal (Refereed)
    Abstract [en]

    In this paper, multivariate calibration of complicated process fluorescence data is presented. Two data sets related to the production of white sugar are investigated. The first data set comprises 106 observations and 571 spectral variables, and the second data set 268 observations and 3997 spectral variables. In both applications, a single response, ash content, is modelled and predicted as a function of the spectral variables. Both data sets contain certain features making multivariate calibration efforts non-trivial. The objective is to show how principal component analysis (PCA) and partial least squares (PLS) regression can be used to overview the data sets and to establish predictively sound regression models. It is shown how a recently developed technique for signal filtering, orthogonal signal correction (OSC), can be applied in multivariate calibration to enhance predictive power. In addition, signal compression is tested on the larger data set using wavelet analysis. It is demonstrated that a compression down to 4% of the original matrix size - in the variable direction - is possible without loss of predictive power. It is concluded that the combination of OSC for pre-processing and wavelet analysis for compression of spectral data is promising for future use.

  • 191.
    Eriksson, Therese
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Varshney, Gaurav
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Aspenström, Pontus
    Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden.
    Palmer, Ruth
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Characterization of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster2010In: BMC Developmental Biology, ISSN 1471-213X, E-ISSN 1471-213X, Vol. 10, no 86Article in journal (Refereed)
    Abstract [en]

    Background: In Drosophila muscle cell fusion takes place both during the formation of the somatic mesodermand the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The coreprocess of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin actsby binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. WhileVerprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceralmuscle fusion has not been determined.Results: Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggestinga role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceralmesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre,which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse,indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells arecapable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of theVerprolin mutant phenotype.Conclusions: Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shownby mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion ineither the visceral or the somatic mesoderm.

  • 192.
    Eriksson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Varshney, Gaurav
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aspenström, Pontus
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster2010In: BMC Developmental Biology, ISSN 1471-213X, E-ISSN 1471-213X, Vol. 10, p. 86-Article in journal (Refereed)
    Abstract [en]

    Background

    In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.

    Results

    Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype.

    Conclusions

    Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm.

  • 193.
    Esberg, Anders
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Moqtaderi, Zarmik
    Fan, Xiaochun
    Lu, Jian
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Struhl, Kevin
    Byström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Iwr1 protein is important for preinitiation complex formation by all three nuclear RNA polymerases in Saccharomyces cerevisiae2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 6, p. e20829-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Iwr1, a protein conserved throughout eukaryotes, was originally identified by its physical interaction with RNA polymerase (Pol) II.

    PRINCIPAL FINDINGS: Here, we identify Iwr1 in a genetic screen designed to uncover proteins involved in Pol III transcription in S. cerevisiae. Iwr1 is important for Pol III transcription, because an iwr1 mutant strain shows reduced association of TBP and Pol III at Pol III promoters, a decreased rate of Pol III transcription, and lower steady-state levels of Pol III transcripts. Interestingly, an iwr1 mutant strain also displays reduced association of TBP to Pol I-transcribed genes and of both TBP and Pol II to Pol II-transcribed promoters. Despite this, rRNA and mRNA levels are virtually unaffected, suggesting a post-transcriptional mechanism compensating for the occupancy defect.

    CONCLUSIONS: Thus, Iwr1 plays an important role in preinitiation complex formation by all three nuclear RNA polymerases.

  • 194.
    Escamez, Sacha
    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).
    Latha Gandla, Madhavi
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Derba-Maceluch, Marta
    Lundqvist, Sven-Olof
    Mellerowicz, Ewa J.
    Jönsson, Leif J.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tuominen, Hannele
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    A collection of genetically engineered Populus trees reveals wood biomass traits that predict glucose yield from enzymatic hydrolysis2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 15798Article in journal (Refereed)
    Abstract [en]

    Wood represents a promising source of sugars to produce bio-based renewables, including biofuels. However, breaking down lignocellulose requires costly pretreatments because lignocellulose is recalcitrant to enzymatic saccharification. Increasing saccharification potential would greatly contribute to make wood a competitive alternative to petroleum, but this requires improving wood properties. To identify wood biomass traits associated with saccharification, we analyzed a total of 65 traits related to wood chemistry, anatomy and structure, biomass production and saccharification in 40 genetically engineered Populus tree lines. These lines exhibited broad variation in quantitative traits, allowing for multivariate analyses and mathematical modeling. Modeling revealed that seven wood biomass traits associated in a predictive manner with saccharification of glucose after pretreatment. Four of these seven traits were also negatively associated with biomass production, suggesting a trade-off between saccharification potential and total biomass, which has previously been observed to offset the overall sugar yield from whole trees. We therefore estimated the "total-wood glucose yield" (TWG) from whole trees and found 22 biomass traits predictive of TWG after pretreatment. Both saccharification and TWG were associated with low abundant, often overlooked matrix polysaccharides such as arabinose and rhamnose which possibly represent new markers for improved Populus feedstocks.

  • 195.
    Espaillat, Akbar
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Carrasco-Lopez, Cesar
    Bernardo-Garcia, Noelia
    Pietrosemoli, Natalia
    Otero, Lisandro H.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A.
    Pazos, Florencio
    Davis, Brigid M.
    Waldor, Matthew K.
    Hermoso, Juan A.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Structural basis for the broad specificity of a new family of amino-acid racemases2014In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 70, p. 79-90Article in journal (Refereed)
    Abstract [en]

    Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

  • 196.
    Espanha, Maria
    et al.
    Faculty of Human Kinetics, Technical University of Lisboa, Estrada da Costa, Cruz Quehrada, Lishoa, Portugal.
    Lammi, Pirkko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Hyttinen, Mika
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Helminen, Heikki
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Extracellular matrix composition of full-thickness defect repair tissue is little influenced by exercise in rat articular cartilage.2001In: Connective Tissue Research, ISSN 0300-8207, E-ISSN 1607-8438, Vol. 42, no 2, p. 97-109, article id 11718471Article in journal (Refereed)
    Abstract [en]

    Full-thickness articular cartilage defects in the femoral condyles of adult rats were examined four and eight weeks after injury. Quantitative polarized light microscopic analysis showed that birefringence of the tissue in the central repair area increased more in rats exercised on a treadmill. Glycosaminoglycan content in the repair tissue was also higher than in the intermittent active motion group at four weeks after injury, but by eight weeks the levels were similar in both groups. No normal-looking articular cartilage was formed in the lesions, and only in one animal type II collagen was observed in the superficial zone of repair tissue. No 3B3(-) antigenicity of the proteoglycans was seen during repair. In conclusion, exercise minimally modified the repair of full-thickness articular cartilage defects in adult rats. The repair in the exercised group may occur slightly faster in the early stages but no difference was seen at the eight week time interval between the exercised and the intermittently active group.

  • 197. Eta, Valerie
    et al.
    Maki-Arvela, Paivi
    Salminen, Eero
    Salmi, Tapio
    Murzin, Dmitry Yu.
    Mikkola, Jyri-Pekka
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The Effect of Alkoxide Ionic Liquids on the Synthesis of Dimethyl Carbonate from CO2 and Methanol over ZrO2-MgO2011In: Catalysis Letters, ISSN 1011-372X, E-ISSN 1572-879X, Vol. 141, no 9, p. 1254-1261Article in journal (Refereed)
    Abstract [en]

    The use of carbon dioxide in the synthesis of chemicals, such as dimethyl carbonate (DMC), constitutes an environmentally attractive alternative to hazardous and toxic reagents. However, the direct synthesis of DMC from methanol and CO2 is characterized by low yields due to the reaction equilibrium and the thermodynamic limitations (Delta G(298K)(0) = + 26.3 kj/mol). Alkoxide ionic liquids possessing alkylimidazolium and benzalkonium cations were prepared, characterised and tested together with ZrO2-MgO catalyst for the synthesis of DMC from methanol and CO2. By using the novel ionic liquid as additives, ca. 12% conversion of methanol, and 90% selectivity to DMC was obtained at 120 A degrees C and 7.5 MPa. The water abstracting potential of the ionic liquids influenced the conversion of methanol and the selectivity to DMC. The alkoxide ionic liquids were recovered and reused in DMC synthesis without loss in activity and selectivity.

  • 198. Fabrik, Ivo
    et al.
    Link, Marek
    Härtlova, Anetta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Dankova, Vera
    Rehulka, Pavel
    Stulik, Jiri
    Application of SILAC labeling to primary bone marrow-derived dendritic cells reveals extensive GM-CSF-dependent arginine metabolism2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 2, p. 752-762Article in journal (Refereed)
    Abstract [en]

    Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the into the field of DC research would therefore be highly beneficial. quantitative proteomic technique of metabolic labeling (SILAC) To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of C-13/N-15-labeled amino acids.. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, similar to 20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of N-15 from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of >= 90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.

  • 199. Falahati, Hanieh
    et al.
    Pazhang, Mohammad
    Zareian, Shekufeh
    Ghaemi, Nasser
    Rofougaran, Reza
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rezaie, Alireza R
    Khajeh, Khosro
    Transmitting the allosteric signal in methylglyoxal synthase2013In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 26, no 7, p. 445-452Article in journal (Refereed)
    Abstract [en]

    The homohexameric enzyme methylglyoxal synthase (MGS) converts dihydroxyacetone phosphate (DHAP) to methylglyoxal and phosphate. This enzyme is allosterically inhibited by phosphate. The allosteric signal induced by phosphate in MGS from Thermus sp. GH5 (TMGS) has been tracked by site-directed mutagenesis, from the binding site of phosphate to the pathways that transmit the signal, and finally to the active site which is the receiver of the signal. In TMGS, Ser-55 distinguishes the inhibitory phosphate from the phosphoryl group of the substrate, DHAP, and transmits the allosteric signal through Pro-82, Arg-97 and Val-101 to the active site. Furthermore, the addition of a C-terminal tail to TMGS reinforces the allosteric signal by introducing a new salt bridge between Asp-10 and an Arg in this tail. Lastly, the active site amino acid, Gly-56, is shown to be involved in both allostery and phosphate elimination step from DHAP by TMGS. Interestingly, some of the mutations also trigger homotropic allostery, supporting the hypothesis that allostery is an intrinsic property of all dynamic proteins. The details of the TMGS allosteric network discussed in this study can serve as a model system for understanding the enigmatic allosteric mechanism of other proteins.

  • 200. Farkas, Sanja A
    et al.
    Böttiger, Anna K
    Isaksson, Helena S
    Finnell, Richard H
    Ren, Aiguo
    Nilsson, Torbjörn K
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry. Örebro Univ Hosp, Dept Lab Med, Örebro, Sweden.
    Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia2013In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 8, no 3, p. 303-316Article in journal (Refereed)
    Abstract [en]

    The objectives of this study were to identify tissue-specific differentially methylated regions (T-DMR's) in the folate transport genes in placental tissue compared with leukocytes, and from placental tissues obtained from normal infants or with neural tube defects (NTDs). Using pyrosequencing, we developed methylation assays for the CpG islands (CGIs) and the CGI shore regions of the folate receptor α (FOLR1), proton-coupled folate transporter (PCFT) and reduced folate carrier 1 (RFC1) genes. The T-DMRs differed in location for each gene and the difference in methylation ranged between 2 and 54%. A higher T-DMR methylated fraction was associated with a lower mRNA level of the FOLR1 and RFC1 genes. Methylation fractions differed according to RFC1 80G > A genotype in the NTD cases and in leukocytes from subjects with high total plasma homocysteine (tHcy). There were no differences in methylated fraction of folate transporter genes between NTD cases and controls. We suggest that T-DMRs participate in the regulation of expression of the FOLR1 and RFC1 genes, that the RFC1 80G > A polymorphism exerts a gene-nutrition interaction on DNA methylation in the RFC1 gene, and that this interaction appears to be most prominent in NTD-affected births and in subjects with high tHcy concentrations.

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