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  • 101.
    Andersson, Jan O
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Andersson, Siv GE
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    A century of typhus, lice and Rickettsia2000In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 151, no 2, p. 143-150Article in journal (Refereed)
    Abstract [en]

    At the beginning of the 20th century, it was discovered at the Pasteur Institute in Tunis that epidemic typhus is transmitted by the human body louse. The complete genome sequence of its causative agent, Rickettsia prowazekii, was determined at Uppsala University in Sweden at the end of the century. In this mini-review, we discuss insights gained from the genome sequence of this fascinating and deadly organism.

  • 102.
    Andersson, Jan O
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Siv GE
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Genomic rearrangements during evolution of the obligate intracellular parasite Rickettsia prowazekii as inferred from an analysis of 52015 bp nucleotide sequence1997In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 143, no 8, p. 2783-2795Article in journal (Other academic)
    Abstract [en]

    In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome of Rickettsia prowazekii, a member of the alpha-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue of metK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures of Escherichia coli, Haemophilus influenzae and Bacillus subtilis have revealed several atypical gene organization patterns in the R. prowazekii genome. For example, R. prowazekii was found to have a unique arrangement of genes upstream of dnaA in a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that the R. prowazekii genome is a highly derived genome and provide examples of gene order structures that are unique for the Rickettsia.

  • 103.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hirt, Robert P.
    Foster, Peter G.
    Roger, Andrew J.
    Evolution of four gene families with patchy phylogenetic distributions: influx of genes into protist genomes2006In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 6, article id 27Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses. RESULTS: The addition of homologs from phagotrophic protists, including several Entamoeba species, the pelobiont Mastigamoeba balamuthi, and the parabasalid Trichomonas vaginalis, and a large quantity of sequences from genome projects resulted in an apparent increase in the number of putative transfer events affecting all three domains of life. Some of the eukaryotic transfers affect a wide range of protists, such as three divergent lineages of Amoebozoa, represented by Entamoeba, Mastigamoeba, and Dictyostelium, while other transfers only affect a limited diversity, for example only the Entamoeba lineage. These observations are consistent with a model where these genes have been introduced into protist genomes independently from various sources over a long evolutionary time. CONCLUSION: Phylogenetic analyses of the updated datasets using more sophisticated phylogenetic methods, in combination with the gene distribution analyses, strengthened, rather than weakened, the support for LGT as an important mechanism affecting the evolution of these gene families. Thus, gene transfer seems to be an on-going evolutionary mechanism by which genes are spread between unrelated lineages of all three domains of life, further indicating the importance of LGT from non-organellar sources into eukaryotic genomes.

  • 104.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organism Biology, Molecular Evolution.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The genome of Giardia and other diplomonads2010In: Anaerobic Parasitic Protozoa: Genomics and Molecular Biology / [ed] C. Graham Clark, Patricia J. Johnson, Rodney D. Adam, Caister Academic Press , 2010, p. 23-44Chapter in book (Other academic)
  • 105.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Roger, Andrew J.
    Evolution of glutamate dehydrogenase genes: evidence for lateral gene transfer within and between prokaryotes and eukaryotes2003In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 3, p. 14-Article in journal (Refereed)
    Abstract [en]

    Background

    Lateral gene transfer can introduce genes with novel functions into genomes or replace genes with functionally similar orthologs or paralogs. Here we present a study of the occurrence of the latter gene replacement phenomenon in the four gene families encoding different classes of glutamate dehydrogenase (GDH), to evaluate and compare the patterns and rates of lateral gene transfer (LGT) in prokaryotes and eukaryotes.

    Results

    We extend the taxon sampling of gdh genes with nine new eukaryotic sequences and examine the phylogenetic distribution pattern of the various GDH classes in combination with maximum likelihood phylogenetic analyses. The distribution pattern analyses indicate that LGT has played a significant role in the evolution of the four gdh gene families. Indeed, a number of gene transfer events are identified by phylogenetic analyses, including numerous prokaryotic intra-domain transfers, some prokaryotic inter-domain transfers and several inter-domain transfers between prokaryotes and microbial eukaryotes (protists).

    Conclusion

    LGT has apparently affected eukaryotes and prokaryotes to a similar extent within the gdh gene families. In the absence of indications that the evolution of the gdh gene families is radically different from other families, these results suggest that gene transfer might be an important evolutionary mechanism in microbial eukaryote genome evolution.

  • 106.
    Andersson, Jan O
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Mikrobiologi.
    Sarchfield, Stewart W
    Roger, Andrew J
    Gene transfers from nanoarchaeota to an ancestor of diplomonads and parabasalids.2005In: Mol Biol Evol, ISSN 0737-4038, Vol. 22, no 1, p. 85-90Article in journal (Refereed)
    Abstract [en]

    Rare evolutionary events, such as lateral gene transfers and gene fusions, may be useful to pinpoint, and correlate the timing of, key branches across the tree of life. For example, the shared possession of a transferred gene indicates a phylogenetic relationship among organismal lineages by virtue of their shared common ancestral recipient. Here, we present phylogenetic analyses of prolyl-tRNA and alanyl-tRNA synthetase genes that indicate lateral gene transfer events to an ancestor of the diplomonads and parabasalids from lineages more closely related to the newly discovered archaeal hyperthermophile Nanoarchaeum equitans (Nanoarchaeota) than to Crenarchaeota or Euryarchaeota. The support for this scenario is strong from all applied phylogenetic methods for the alanyl-tRNA sequences, whereas the phylogenetic analyses of the prolyl-tRNA sequences show some disagreements between methods, indicating that the donor lineage cannot be identified with a high degree of certainty. However, in both trees, the diplomonads and parabasalids branch together within the Archaea, strongly suggesting that these two groups of unicellular eukaryotes, often regarded as the two earliest independent offshoots of the eukaryotic lineage, share a common ancestor to the exclusion of the eukaryotic root. Unfortunately, the phylogenetic analyses of these two aminoacyl-tRNA synthetase genes are inconclusive regarding the position of the diplomonad/parabasalid group within the eukaryotes. Our results also show that the lineage leading to Nanoarchaeota branched off from Euryarchaeota and Crenarchaeota before the divergence of diplomonads and parabasalids, that this unexplored archaeal diversity, currently only represented by the hyperthermophilic organism Nanoarchaeum equitans, may include members living in close proximity to mesophilic eukaryotes, and that the presence of split genes in the Nanoarchaeum genome is a derived feature.

  • 107.
    Andersson, Jan O.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Sjögren, Åsa M.
    Horner, David S.
    Murphy, Colleen A.
    Dyal, Patricia L.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Logsdon, Jr., John M.
    Ragan, Mark A.
    Hirt, Robert P.
    Roger, Andrew J.
    A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution2007In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 8, p. 51-Article, review/survey (Refereed)
    Abstract [en]

    Background: Comparative genomic studies of the mitochondrion-lacking protist group Diplomonadida (diplomonads) has been lacking, although Giardia lamblia has been intensively studied. We have performed a sequence survey project resulting in 2341 expressed sequence tags (EST) corresponding to 853 unique clones, 5275 genome survey sequences (GSS), and eleven finished contigs from the diplomonad fish parasite Spironucleus salmonicida (previously described as S. barkhanus). Results: The analyses revealed a compact genome with few, if any, introns and very short 3′ untranslated regions. Strikingly different patterns of codon usage were observed in genes corresponding to frequently sampled ESTs versus genes poorly sampled, indicating that translational selection is influencing the codon usage of highly expressed genes. Rigorous phylogenomic analyses identified 84 genes - mostly encoding metabolic proteins - that have been acquired by diplomonads or their relatively close ancestors via lateral gene transfer (LGT). Although most acquisitions were from prokaryotes, more than a dozen represent likely transfers of genes between eukaryotic lineages. Many genes that provide novel insights into the genetic basis of the biology and pathogenicity of this parasitic protist were identified including 149 that putatively encode variant-surface cysteine-rich proteins which are candidate virulence factors. A number of genomic properties that distinguish S. salmonicida from its human parasitic relative G. lamblia were identified such as nineteen putative lineage-specific gene acquisitions, distinct mutational biases and codon usage and distinct polyadenylation signals. Conclusion: Our results highlight the power of comparative genomic studies to yield insights into the biology of parasitic protists and the evolution of their genomes, and suggest that genetic exchange between distantly-related protist lineages may be occurring at an appreciable rate in eukaryote genome evolution.

  • 108. Andersson, Magnus
    et al.
    Vincent, Jonathan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Davidsson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science.
    Neutze, Richard
    A proposed time-resolved X-ray scattering approach to track local and global conformational changes in membrane transport proteins2008In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 16, no 1, p. 21-28Article in journal (Refereed)
    Abstract [en]

    Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed by halorhodopsin as a model system, we combine molecular dynamics simulations with X-ray scattering calculations to demonstrate how small-molecule time-resolved X-ray scattering can be extended to the study of membrane transport processes. In particular, by introducing strongly scattering atoms to label specific positions within the protein and substrate, the technique of time-resolved wide-angle X-ray scattering can reveal both local and global conformational changes. This approach simultaneously enables the direct visualization of global rearrangements and substrate movement, crucial concepts that underpin the alternating access paradigm for membrane transport proteins.

  • 109. Andersson, Marlene
    et al.
    Chen, Gefei
    Otikovs, Martins
    Landreh, Michael
    Nordling, Kerstin
    Kronqvist, Nina
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Jornvall, Hans
    Knight, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ridderstrale, Yvonne
    Holm, Lena
    Meng, Qing
    Jaudzems, Kristaps
    Chesler, Mitchell
    Johansson, Jan
    Rising, Anna
    Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains2014In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, no 8, p. e1001921-Article in journal (Refereed)
    Abstract [en]

    Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

  • 110. Andersson, Mattias
    et al.
    Gallwitz, Maike
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Enoksson, Mattias
    Hellman, Lars
    The extended substrate specificity of the human chymase reveals a serine protease with well-defined substrate recognition profileManuscript (Other academic)
  • 111.
    Andersson, Mattias K.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cleavage Specificity of Mast Cell Chymases2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents.

    As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics.

    The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential in vivo substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme.

    Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference.

    Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.

    List of papers
    1. Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse - High specificity indicates high substrate selectivity
    Open this publication in new window or tab >>Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse - High specificity indicates high substrate selectivity
    2008 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 9, p. 2548-2558Article in journal (Refereed) Published
    Abstract [en]

    Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.

    Keywords
    Mast cell, Chymase, Mouse mast cell protease-1, Mouse mast cell protease-2, Cleavage specificity, Mucosal immunity, Intestinal permeability
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-97162 (URN)10.1016/j.molimm.2008.01.012 (DOI)000255528400013 ()18313755 (PubMedID)
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
    2. The extended cleavage specificity of the rodent β-chymases rMCP-1 and mMCP-4 reveal major fumctional similarities to the human mast cell chymase
    Open this publication in new window or tab >>The extended cleavage specificity of the rodent β-chymases rMCP-1 and mMCP-4 reveal major fumctional similarities to the human mast cell chymase
    2008 (English)In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 3, p. 766-775Article in journal (Refereed) Published
    Abstract [en]

    In rat and mouse the phylogenetic homologues of the human mast cell alpha-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate alpha-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell beta-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1' and P3'. The two enzymes differ clearly only in one position, the P2' residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2' of known substrates for the human chymase. Overall, these two rodent beta-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.

    Keywords
    Mast cell, Chymase, Rat mast cell protease-1, Mouse mast cell protease-4, Cleavage specificity
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-97163 (URN)10.1016/j.molimm.2007.06.360 (DOI)000251350900020 ()17681377 (PubMedID)
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
    3. The extended substrate cleavage specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile
    Open this publication in new window or tab >>The extended substrate cleavage specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile
    2009 (English)In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 21, no 1, p. 95-104Article in journal (Refereed) Published
    Abstract [en]

    The human chymase (HC) is a major granule constituent of mast cells (MCs) residing in the connective tissue and the sub-mucosa. Although many potential substrates have been described for this important MC enzyme, its full range of in vivo substrates has most likely not yet been identified. A major step toward a better understanding of the function of the HC is therefore to determine its extended cleavage specificity. Using a phage-displayed random nonapeptide library, we show that the HC has a rather stringent substrate recognition profile. Only aromatic amino acids (aa) are accepted in position P1, with a   strong preference for Tyr and Phe over Trp. Aliphatic aa are preferred in positions P2 to P4 N-terminal of the cleaved bond. In the P1' position C-terminal of the cleaved bond, Ser is clearly over-represented and acidic aa Asp and Glu are strongly preferred in the P2' position. In P3', the small aliphatic aa Ala, Val and Gly were frequently observed. The consensus sequence, from P4 to P3': Gly/Leu/Val-Val/Ala/Leu-Ala/Val/Leu-Tyr/Phe-Ser-Asp/Glu-Ala/Val/Gly,   provides an instrument for the identification of novel in vivo substrates for the HC. Interestingly, a very similar cleavage specificity was recently reported for the major chymase in mouse connective tissue mast cells (CTMCs), the beta-chymase mouse mast cell   protease-4, suggesting functional homology between these two enzymes. This indicates that a rather stringent chymotryptic substrate recognition profile has been evolutionary conserved for the dominant CTMC chymase in mammals.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-97164 (URN)10.1093/intimm/dxn128 (DOI)000261898300009 ()
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
    4. Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
    Open this publication in new window or tab >>Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates
    2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 10, p. 2255-2267Article in journal (Refereed) Published
    Abstract [en]

    Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-97165 (URN)10.1111/j.1742-4658.2010.07642.x (DOI)000277084600007 ()
    Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
  • 112.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Enoksson, Mattias
    Gallwitz, Maike
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The extended substrate cleavage specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile2009In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 21, no 1, p. 95-104Article in journal (Refereed)
    Abstract [en]

    The human chymase (HC) is a major granule constituent of mast cells (MCs) residing in the connective tissue and the sub-mucosa. Although many potential substrates have been described for this important MC enzyme, its full range of in vivo substrates has most likely not yet been identified. A major step toward a better understanding of the function of the HC is therefore to determine its extended cleavage specificity. Using a phage-displayed random nonapeptide library, we show that the HC has a rather stringent substrate recognition profile. Only aromatic amino acids (aa) are accepted in position P1, with a   strong preference for Tyr and Phe over Trp. Aliphatic aa are preferred in positions P2 to P4 N-terminal of the cleaved bond. In the P1' position C-terminal of the cleaved bond, Ser is clearly over-represented and acidic aa Asp and Glu are strongly preferred in the P2' position. In P3', the small aliphatic aa Ala, Val and Gly were frequently observed. The consensus sequence, from P4 to P3': Gly/Leu/Val-Val/Ala/Leu-Ala/Val/Leu-Tyr/Phe-Ser-Asp/Glu-Ala/Val/Gly,   provides an instrument for the identification of novel in vivo substrates for the HC. Interestingly, a very similar cleavage specificity was recently reported for the major chymase in mouse connective tissue mast cells (CTMCs), the beta-chymase mouse mast cell   protease-4, suggesting functional homology between these two enzymes. This indicates that a rather stringent chymotryptic substrate recognition profile has been evolutionary conserved for the dominant CTMC chymase in mammals.

  • 113.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Karlson, Ulrika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The extended cleavage specificity of the rodent β-chymases rMCP-1 and mMCP-4 reveal major fumctional similarities to the human mast cell chymase2008In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 3, p. 766-775Article in journal (Refereed)
    Abstract [en]

    In rat and mouse the phylogenetic homologues of the human mast cell alpha-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate alpha-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell beta-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1' and P3'. The two enzymes differ clearly only in one position, the P2' residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2' of known substrates for the human chymase. Overall, these two rodent beta-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.

  • 114.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Immunology.
    Pemberton, Alan D.
    Miller, Hugh R. P.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Immunology.
    Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse - High specificity indicates high substrate selectivity2008In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, no 9, p. 2548-2558Article in journal (Refereed)
    Abstract [en]

    Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.

  • 115.
    Andersson, Mattias K.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Thorpe, Michael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Chemical Biology.
    Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2′ of substrates2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 10, p. 2255-2267Article in journal (Refereed)
    Abstract [en]

    Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143 -> Gln and Lys192 -> Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

  • 116.
    Andersson, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Decoding the Structural Layer of Transcriptional Regulation: Computational Analyses of Chromatin and Chromosomal Aberrations2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Gene activity is regulated at two separate layers. Through structural and chemical properties of DNA – the primary layer of encoding – local signatures may enable, or disable, the binding of proteins or complexes of them with regulatory potential to the DNA. At a higher level – the structural layer of encoding – gene activity is regulated through the properties of higher order DNA structure, chromatin, and chromosome organization. Cells with abnormal chromosome compaction or organization, e.g. cancer cells, may thus have perturbed regulatory activities resulting in abnormal gene activity.

    Hence, there is a great need to decode the transcriptional regulation encoded in both layers to further our understanding of the factors that control activity and life of a cell and, ultimately, an organism. Modern genome-wide studies with those aims rely on data-intense experiments requiring sophisticated computational and statistical methods for data handling and analyses. This thesis describes recent advances of analyzing experimental data from quantitative biological studies to decipher the structural layer of encoding in human cells.

    Adopting an integrative approach when possible, combining multiple sources of data, allowed us to study the influences of chromatin (Papers I and II) and chromosomal aberrations (Paper IV) on transcription. Combining chromatin data with chromosomal aberration data allowed us to identify putative driver oncogenes and tumor-suppressor genes in cancer (Paper IV).

    Bayesian approaches enabling the incorporation of background information in the models and the adaptability of such models to data have been very useful. Their usages yielded accurate and narrow detection of chromosomal breakpoints in cancer (Papers III and IV) and reliable positioning of nucleosomes and their dynamics during transcriptional regulation at functionally relevant regulatory elements (Paper II).

    Using massively parallel sequencing data, we explored the chromatin landscapes of human cells (Papers I and II) and concluded that there is a preferential and evolutionary conserved positioning at internal exons nearly unaffected by the transcriptional level. We also observed a strong association between certain histone modifications and the inclusion or exclusion of an exon in the mature gene transcript, suggesting a functional role in splicing.

    List of papers
    1. Nucleosomes are well positioned in exons and carry characteristic histone modifications
    Open this publication in new window or tab >>Nucleosomes are well positioned in exons and carry characteristic histone modifications
    Show others...
    2009 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 19, no 10, p. 1732-1741Article in journal (Refereed) Published
    Abstract [en]

    The genomes of higher organisms are packaged in nucleosomes with functional histone modifications. Until now, genome-wide nucleosome and histone modification studies have focused on transcription start sites (TSSs) where nucleosomes in RNA polymerase II (RNAPII) occupied genes are well positioned and have histone modifications that are characteristic of expression status. Using public data, we here show that there is a higher nucleosome-positioning signal in internal human exons and that this positioning is independent of expression. We observed a similarly strong nucleosome-positioning signal in internal exons of C. elegans. Among the 38 histone modifications analyzed in man, H3K36me3, H3K79me1, H2BK5me1, H3K27me1, H3K27me2 and H3K27me3 had evidently higher signal in internal exons than in the following introns and were clearly related to exon expression. These observations are suggestive of roles in splicing. Thus, exons are not only characterized by their coding capacity but also by their nucleosome organization, which seems evolutionary conserved since it is present in both primates and nematodes.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-107609 (URN)10.1101/gr.092353.109 (DOI)000270389700005 ()19687145 (PubMedID)
    Note

    De tre första författarna delar första författarskapet.

    Available from: 2009-08-19 Created: 2009-08-19 Last updated: 2017-12-13Bibliographically approved
    2. Strand-based mixture modeling of nucleosome positioning in HepG2 cells and their regulatory dynamics in response to TGF-beta treatment
    Open this publication in new window or tab >>Strand-based mixture modeling of nucleosome positioning in HepG2 cells and their regulatory dynamics in response to TGF-beta treatment
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-130998 (URN)
    Available from: 2010-09-20 Created: 2010-09-20 Last updated: 2010-11-11
    3. A Segmental Maximum A Posteriori Approach to Genome-wide Copy Number Profiling
    Open this publication in new window or tab >>A Segmental Maximum A Posteriori Approach to Genome-wide Copy Number Profiling
    Show others...
    2008 (English)In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 6, p. 751-758Article in journal (Other academic) Published
    Abstract [en]

    MOTIVATION: Copy number profiling methods aim at assigning DNA copy numbers to chromosomal regions using measurements from microarray-based comparative genomic hybridizations. Among the proposed methods to this end, Hidden Markov Model (HMM)-based approaches seem promising since DNA copy number transitions are naturally captured in the model. Current discrete-index HMM-based approaches do not, however, take into account heterogeneous information regarding the genomic overlap between clones. Moreover, the majority of existing methods are restricted to chromosome-wise analysis. RESULTS: We introduce a novel Segmental Maximum A Posteriori approach, SMAP, for DNA copy number profiling. Our method is based on discrete-index Hidden Markov Modeling and incorporates genomic distance and overlap between clones. We exploit a priori information through user-controllable parameterization that enables the identification of copy number deviations of various lengths and amplitudes. The model parameters may be inferred at a genome-wide scale to avoid overfitting of model parameters often resulting from chromosome-wise model inference. We report superior performances of SMAP on synthetic data when compared with two recent methods. When applied on our new experimental data, SMAP readily recognizes already known genetic aberrations including both large-scale regions with aberrant DNA copy number and changes affecting only single features on the array. We highlight the differences between the prediction of SMAP and the compared methods and show that SMAP accurately determines copy number changes and benefits from overlap consideration.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-13616 (URN)10.1093/bioinformatics/btn003 (DOI)000254010400003 ()18204059 (PubMedID)
    Available from: 2008-08-21 Created: 2008-08-21 Last updated: 2017-12-11Bibliographically approved
    4. Integrative epigenomic and genomic analysis of malignant pheochromocytoma
    Open this publication in new window or tab >>Integrative epigenomic and genomic analysis of malignant pheochromocytoma
    Show others...
    2010 (English)In: Experimental and Molecular Medicine, ISSN 1226-3613, E-ISSN 2092-6413, Vol. 42, no 7, p. 484-502Article in journal (Refereed) Published
    Abstract [en]

    Epigenomic and genomic changes affect gene expression and contribute to tumor development. The histone modifications trimethylated histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) are epigenetic regulators associated to active and silenced genes, respectively and alterations of these modifications have been observed in cancer. Furthermore, genomic aberrations such as DNA copy number changes are common events in tumors. Pheochromocytoma is a rare endocrine tumor of the adrenal gland that mostly occurs sporadic with unknown epigenetic/genetic cause. The majority of cases are benign. Here we aimed to combine the genome-wide profiling of H3K4me3 and H3K27me3, obtained by the ChIP-chip methodology, and DNA copy number data with global gene expression examination in a malignant pheochromocytoma sample. The integrated analysis of the tumor expression levels, in relation to normal adrenal medulla, indicated that either histone modifications or chromosomal alterations, or both, have great impact on the expression of a substantial fraction of the genes in the investigated sample. Candidate tumor suppressor genes identified with decreased expression, a H3K27me3 mark and/or in regions of deletion were for instance TGIF1, DSC3, TNFRSF10B, RASSF2, HOXA9, PTPRE and CDH11. More genes were found with increased expression, a H3K4me3 mark, and/or in regions of gain. Potential oncogenes detected among those were GNAS, INSM1, DOK5, ETV1, RET, NTRK1, IGF2, and the H3K27 trimethylase gene EZH2. Our approach to associate histone methylations and DNA copy number changes to gene expression revealed apparent impact on global gene transcription, and enabled the identification of candidate tumor genes for further exploration.

    Keywords
    histone code, DNA copy number changes, gene expression, oncogenes, pheochromocytoma, tumor suppressor genes
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-129532 (URN)10.3858/emm.2010.42.7.050 (DOI)000280558100002 ()20534969 (PubMedID)
    Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2017-12-12Bibliographically approved
  • 117.
    Andersson, Robin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Bruder, Carl E G
    Piotrowski, Arkadiusz
    Menzel, Uwe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nord, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sandgren, Johanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Hvidsten, Torgeir R
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    de Ståhl, Teresita Diaz
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dumanski, Jan P
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    A Segmental Maximum A Posteriori Approach to Genome-wide Copy Number Profiling2008In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 6, p. 751-758Article in journal (Other academic)
    Abstract [en]

    MOTIVATION: Copy number profiling methods aim at assigning DNA copy numbers to chromosomal regions using measurements from microarray-based comparative genomic hybridizations. Among the proposed methods to this end, Hidden Markov Model (HMM)-based approaches seem promising since DNA copy number transitions are naturally captured in the model. Current discrete-index HMM-based approaches do not, however, take into account heterogeneous information regarding the genomic overlap between clones. Moreover, the majority of existing methods are restricted to chromosome-wise analysis. RESULTS: We introduce a novel Segmental Maximum A Posteriori approach, SMAP, for DNA copy number profiling. Our method is based on discrete-index Hidden Markov Modeling and incorporates genomic distance and overlap between clones. We exploit a priori information through user-controllable parameterization that enables the identification of copy number deviations of various lengths and amplitudes. The model parameters may be inferred at a genome-wide scale to avoid overfitting of model parameters often resulting from chromosome-wise model inference. We report superior performances of SMAP on synthetic data when compared with two recent methods. When applied on our new experimental data, SMAP readily recognizes already known genetic aberrations including both large-scale regions with aberrant DNA copy number and changes affecting only single features on the array. We highlight the differences between the prediction of SMAP and the compared methods and show that SMAP accurately determines copy number changes and benefits from overlap consideration.

  • 118.
    Andersson, Robin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Barbacioru, Catalin
    Reddy Bysani, Madhu Sudhan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Tuch, Brian
    Lee, Clarence
    Peckham, Heather
    McKernan, Kevin
    de la Vega, Francisco
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Strand-based mixture modeling of nucleosome positioning in HepG2 cells and their regulatory dynamics in response to TGF-beta treatmentManuscript (preprint) (Other academic)
  • 119.
    Andersson, Robin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Enroth, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Rada-Iglesias, Alvaro
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Komorowski, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, The Linnaeus Centre for Bioinformatics.
    Nucleosomes are well positioned in exons and carry characteristic histone modifications2009In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 19, no 10, p. 1732-1741Article in journal (Refereed)
    Abstract [en]

    The genomes of higher organisms are packaged in nucleosomes with functional histone modifications. Until now, genome-wide nucleosome and histone modification studies have focused on transcription start sites (TSSs) where nucleosomes in RNA polymerase II (RNAPII) occupied genes are well positioned and have histone modifications that are characteristic of expression status. Using public data, we here show that there is a higher nucleosome-positioning signal in internal human exons and that this positioning is independent of expression. We observed a similarly strong nucleosome-positioning signal in internal exons of C. elegans. Among the 38 histone modifications analyzed in man, H3K36me3, H3K79me1, H2BK5me1, H3K27me1, H3K27me2 and H3K27me3 had evidently higher signal in internal exons than in the following introns and were clearly related to exon expression. These observations are suggestive of roles in splicing. Thus, exons are not only characterized by their coding capacity but also by their nucleosome organization, which seems evolutionary conserved since it is present in both primates and nematodes.

  • 120.
    Andersson, Robin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, The Linnaeus Centre for Bioinformatics.
    Vitoria, Aida
    Maluszynski, Jan
    Komorowski, Jan
    RoSy: A Rough Knowledge Base System2005In: Rough Sets, Fuzzy Sets, Data Mining, and Granular Computing: 10th International Conference, RSFDGrC 2005, Regina, Canada, August 31 - September 3, 2005, Proceedings, Part II, 2005, p. 48-58Conference paper (Refereed)
    Abstract [en]

    This paper presents a user-oriented view of RoSy, a Rough Knowledge Base System. The system tackles two problems not fully answered by previous research: the ability to define rough sets in terms of other rough sets and incorporation of domain or expert knowledge. We describe two main components of RoSy: knowledge base creation and query answering. The former allows the user to create a knowledge base of rough concepts and checks that the definitions do not cause what we will call a model failure. The latter gives the user a possibility to query rough concepts defined in the knowledge base. The features of RoSy are described using examples. The system is currently available on a web site for online interactions.

  • 121.
    Andersson, Siv G. E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Stress management strategies in single bacterial cells2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 15, p. 3921-3923Article in journal (Other academic)
  • 122.
    Andersson, Siv G. E.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Goodman, A. L.
    Bacterial genomes: Next generation sequencing technologies for studies of bacterial ecosystems2012In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 15, no 5, p. 603-604Article in journal (Other academic)
  • 123.
    Andersson, Siv GE
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Zomorodipour, A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Andersson, Jan O
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sicheritz-Ponten, T
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Alsmark, UCM
    Uppsala University.
    Podowski, RM
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Näslund, A Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Eriksson, Ann-Sofie
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Winkler, HH
    Kurland, Charles G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The genome sequence of Rickettsia prowazekii and the origin of mitochondria1998In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 396, no 6707, p. 133-140Article in journal (Refereed)
    Abstract [en]

    We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of 'neutralized' genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

  • 124.
    Andreasson, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Holmlund, Joakim
    Rauer, Ralf
    Kaell, Mikael
    Boerjesson, Lars
    Knee, Christopher S.
    Eriksson, Annika K.
    Eriksson, Sten-G.
    Ruebhausen, Michael
    Chaudhury, Rajit P.
    Electron-phonon interactions in perovskites containing Fe and Cr studied by Raman scattering using oxygen-isotope and cation substitution2008In: Physical Review B. Condensed Matter and Materials Physics, ISSN 1098-0121, E-ISSN 1550-235X, Vol. 78, no 23, p. 235103-Article in journal (Refereed)
    Abstract [en]

    We use temperature-dependent inelastic light scattering to study the origin of the strong multiphonon scattering of a local oxygen breathing mode present in the mixed B-site orthorhombic (space group Pnma) perovskite LaFe0.5Cr0.5O3 but absent in isostructural LaFeO3 and LaCrO3. It is seen that the multiphonon scattering is critically sensitive to the presence of both Fe and Cr ions on the B site. These results support our interpretation that the multiphonon scattering is activated by local electron-phonon interactions according to the Franck-Condon picture following an Fe-Cr charge transfer. Further, O-18 substitution is performed on the x=0, 0.04, and 0.5 compounds and clearly shows that all modes appearing above the first-order phonon-scattering region in these compounds originate from higher-order oxygen stretching vibrations. In particular this is the case for the strong second-order scattering dominating the scattering response in LaFeO3. Accordingly we propose that these modes are generated by infrared-active longitudinal optical (IR LO) two-phonon and combination scattering activated by Frohlich interaction. For x=0.02 and 0.04 the characteristic IR LO two-phonon and Franck-Condon multiphonon-scattering profiles mix. We also study the influence of isovalent cation substitution and Sr doping in AFe(0.5)Cr(0.5)O(3) (A=La, Nd, and Gd) and La1-ySryFe0.5Cr0.5O3-delta (y=0, 0.16, and 0.5) on the strong electron-phonon coupling present in LaFe0.5Cr0.5O3. The Franck-Condon effect in LaFe0.5Cr0.5O3, is not significantly affected by isovalent A-site substitution, despite the increasing orthorhombic distortion associated with decreasing A-site ionic radii. On the contrary, aliovalent Sr doping causes a rapid decrease in the Franck-Condon scattering. This shows that the strong electron-phonon coupling in these compounds is highly sensitive to local lattice and electronic decoherence but insensitive to global lattice distortions. Finally, a preliminary assignment of the A(g) and B-2g phonon modes in AFe(0.5)Cr(0.5)O(3) (A=La, Nd, and Gd) is made based on the present observations and published results for LaCrO3 and AMnO(3). The modes associated with oxygen octahedral tilt and bending vibrations are heavily influenced by the magnitude of the orthorhombic distortion.

  • 125.
    Andreasson, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Holmlund, Joakim
    Singer, Stefan G.
    Knee, Christopher S.
    Rauer, Ralf
    Schulz, Benjamin
    Kall, Mikael
    Ruebhausen, Michael
    Eriksson, Sten-G
    Börjesson, Lars
    Lichtenstein, Alexander
    Electron-lattice interactions in the perovskite LaFe0.5Cr0.5O3 characterized by optical spectroscopy and LDA plus U calculations2009In: Physical Review B. Condensed Matter and Materials Physics, ISSN 1098-0121, E-ISSN 1550-235X, Vol. 80, no 7, p. 075103-Article in journal (Refereed)
    Abstract [en]

    We use resonance Raman scattering (incident photon energies between 1.8 and 4.13 eV), LDA+U calculations, spectroscopic ellipsometry, and oblique IR reflectivity to characterize the strong electron-phonon interactions in the disordered perovskite LaFe0.5Cr0.5O3. When the photon energy coincides with a Cr to Fe Mott-Hubbard transfer gap around 2.4 eV the electron-phonon interaction is manifested by a Franck-Condon effect with exceptional first-and higher order scattering of a local oxygen breathing mode. At higher incident energies we observe a superposition of Franck-Condon scattering and Frohlich interaction induced infrared active longitudinal optical two-phonon scattering activated mainly by O to Fe charge transfer. Our results establish LaFe0.5Cr0.5O3 as a model compound for research on electron-phonon interactions in strongly correlated complex systems and show that Franck-Condon scattering in complex solids is not limited to Jahn-Teller active compounds.

  • 126.
    Andreasson, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Iwan, Bianca Stella
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Andrejczuk, A.
    Abreu, E.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Bergh, M.
    Caleman, Carl
    Nelson, A. J.
    Bajt, S.
    Chalupsky, J.
    Chapman, H. N.
    Faeustlin, R. R.
    Hajkova, V.
    Heimann, P. A.
    Hjörvarsson, Björgvin
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Physics.
    Juha, L.
    Klinger, D.
    Krzywinski, J.
    Nagler, B.
    Pålsson, Gunnar Karl
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Physics.
    Singer, W.
    Seibert, Marvin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Sobicrajski, R.
    Tolcikis, S.
    Tschentscher, T.
    Vinko, S. M.
    Lee, R. W.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Saturated ablation in metal hydrides and acceleration of protons and deuterons to keV energies with a soft-x-ray laser2011In: Physical Review E. Statistical, Nonlinear, and Soft Matter Physics, ISSN 1539-3755, E-ISSN 1550-2376, Vol. 83, no 1, p. 016403-Article in journal (Refereed)
    Abstract [en]

    Studies of materials under extreme conditions have relevance to a broad area of research, including planetary physics, fusion research, materials science, and structural biology with x-ray lasers. We study such extreme conditions and experimentally probe the interaction between ultrashort soft x-ray pulses and solid targets (metals and their deuterides) at the FLASH free-electron laser where power densities exceeding 1017 W/cm2 were reached. Time-of-flight ion spectrometry and crater analysis were used to characterize the interaction. The results show the onset of saturation in the ablation process at power densities above 1016 W/cm2. This effect can be linked to a transiently induced x-ray transparency in the solid by the femtosecond x-ray pulse at high power densities. The measured kinetic energies of protons and deuterons ejected from the surface reach several keV and concur with predictions from plasma-expansion models. Simulations of the interactions were performed with a nonlocal thermodynamic equilibrium code with radiation transfer. These calculations return critical depths similar to the observed crater depths and capture the transient surface transparency at higher power densities.

  • 127.
    Andreasson, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Martin, Andrew V.
    Liang, Meng
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Aquila, Andrew
    Wang, Fenglin
    Iwan, Bianca
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Svenda, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Ekeberg, Tomas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hantke, Max
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Bielecki, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Rolles, Daniel
    Rudenko, Artem
    Foucar, Lutz
    Hartmann, Robert
    Erk, Benjamin
    Rudek, Benedikt
    Chapman, Henry N.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Barty, Anton
    Automated identification and classification of single particle serial femtosecond X-ray diffraction data2014In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 22, no 3, p. 2497-2510Article in journal (Refereed)
    Abstract [en]

    The first hard X-ray laser, the Linac Coherent Light Source (LCLS), produces 120 shots per second. Particles injected into the X-ray beam are hit randomly and in unknown orientations by the extremely intense X-ray pulses, where the femtosecond-duration X-ray pulses diffract from the sample before the particle structure is significantly changed even though the sample is ultimately destroyed by the deposited X-ray energy. Single particle X-ray diffraction experiments generate data at the FEL repetition rate, resulting in more than 400,000 detector readouts in an hour, the data stream during an experiment contains blank frames mixed with hits on single particles, clusters and contaminants. The diffraction signal is generally weak and it is superimposed on a low but continually fluctuating background signal, originating from photon noise in the beam line and electronic noise from the detector. Meanwhile, explosion of the sample creates fragments with a characteristic signature. Here, we describe methods based on rapid image analysis combined with ion Time-of-Flight (ToF) spectroscopy of the fragments to achieve an efficient, automated and unsupervised sorting of diffraction data. The studies described here form a basis for the development of real-time frame rejection methods, e. g. for the European XFEL, which is expected to produce 100 million pulses per hour. (C)2014 Optical Society of America

  • 128.
    Andreasson, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Timneanu, Nicusor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Iwan, Bianca
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hantke, Max
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Rath, Asawari
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Ekeberg, Tomas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Maia, Filipe R. N. C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Barty, Anton
    Chapman, Henry N.
    Bielecki, Johan
    Abergel, C.
    Seltzer, V.
    Claverie, J.-M.
    Svenda, M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Time of Flight Mass Spectrometry to Monitor Sample Expansion in Flash Diffraction Studies on Single Virus ParticlesManuscript (preprint) (Other academic)
  • 129. Andreev, Dmitri
    et al.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Terenin, Ilya
    Dmitriev, Sergey
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Shatsky, Ivan
    The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome2008In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 14, no 2, p. 233-239Article in journal (Refereed)
    Abstract [en]

    ReIE/ReIB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. ReIE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. ReIE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "ReIE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.

  • 130. Andreev, Dmitri
    et al.
    Hauryliuk, Vasili
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Terenin, Ilya
    Dmitriev, Sergey
    Ehrenberg, Måns
    Shatsky, Ivan
    The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome2008In: RNA, Vol. 14, no 2, p. 233-239Article in journal (Refereed)
  • 131.
    Andresen, Liis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Holmqvist, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala Univ, Biomed Ctr, Dept Cell & Mol Biol, Uppsala, Sweden.
    CLIP-Seq in Bacteria: Global Recognition Patterns of Bacterial RNA-Binding Proteins2018In: High-Density Sequencing Applications in Microbial Molecular Genetics / [ed] Carpousis, A J, ELSEVIER ACADEMIC PRESS INC , 2018, p. 127-145Chapter in book (Refereed)
    Abstract [en]

    RNA-protein interactions are at the heart of many central cellular processes, and RNA-binding proteins (RBPs) associate with virtually all RNA molecules in a cell. In bacteria, global RBPs, often in conjunction with small regulatory RNAs, affect physiology and virulence by controlling transcription, translation, and RNA decay. To understand how these regulatory proteins orchestrate global gene expression, detailed maps of their cellular RNA binding sites are required. To this end, cross-linking and immunoprecipitation followed by deep sequencing (CLIP-seq) has revolutionized RBP studies by providing knowledge about global recognition patterns of RBPs in both eukaryotic and bacterial cells. In this chapter, we provide a step-by-step protocol for global mapping of bona fide RBP binding sites using CLIP-seq in bacteria. This protocol has been successfully applied for charting the binding sites of Hfq, CsrA, and ProQ, three global regulatory RBPs in Salmonella enterica and Escherichia coli, and should be readily applicable to other RBPs and bacterial species.

  • 132.
    Andrén, M
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Xiang, Z
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, G
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Kleinau, S
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    FcgammaRIII-expressing macrophages are essential for development of collagen-induced arthritis.2006In: Scand J Immunol, ISSN 0300-9475, Vol. 63, no 4, p. 282-9Article in journal (Refereed)
  • 133.
    Andrén, Maria
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylär immunologi.
    Johanneson, Bo
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Alarcon Riquelme, Marta E
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Medicinsk Genetik.
    Kleinau, Sandra
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylär immunologi.
    IgG Fc receptor polymorphisms and association with autoimmune disease.2005In: Eur J Immunol, ISSN 0014-2980, Vol. 35, no 10, p. 3020-9Article in journal (Other (popular scientific, debate etc.))
  • 134.
    Andér, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Computational Analysis of Molecular Recognition Involving the Ribosome and a Voltage Gated K+ Channel2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel.

    The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center.

    Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified.

    The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex.

    A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted.

    Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

    List of papers
    1.
    The record could not be found. The reason may be that the record is no longer available or you may have typed in a wrong id in the address field.
    2. Energetics of stop codon recognition on the ribosome
    Open this publication in new window or tab >>Energetics of stop codon recognition on the ribosome
    (English)Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-101412 (URN)
    Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2010-01-14
    3. Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?
    Open this publication in new window or tab >>Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?
    2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 15, p. 3483-3489Article in journal (Refereed) Published
    Abstract [en]

    The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination   reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic   structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is   no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.

    Place, publisher, year, edition, pages
    Washington, DC, USA: American Chemical Society, 2009
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-101411 (URN)10.1021/bi900117r (DOI)000265170200025 ()
    Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2017-12-13Bibliographically approved
    4. Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
    Open this publication in new window or tab >>Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
    2008 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 94, no 3, p. 820-831Article in journal (Refereed) Published
    Abstract [en]

    The binding of blockers to the human voltage-gated Kv1.5 potassium ion channel is investigated using a three-step procedure consisting of homology modeling, automated docking, and binding free energy calculations from molecular dynamics simulations, in combination with the linear interaction energy method. A reliable homology model of Kv1.5 is constructed using the recently published crystal structure of the Kv1.2 channel as a template. This model is expected to be significantly more accurate than earlier ones based on less similar templates. Using the three-dimensional homology model, a series of blockers with known affinities are docked into the cavity of the ion channel and their free energies of binding are calculated. The predicted binding free energies are in very good agreement with experimental data and the binding is predicted to be mainly achieved through nonpolar interactions, whereas the relatively small differences in the polar contribution determine the specificity. Apart from confirming the importance of residues V505, I508, V512, and V516 for ligand binding in the cavity, the results also show that A509 and P513 contribute significantly to the nonpolar binding interactions. Furthermore, we find that pharmacophore models based only on optimized free ligand conformations may not necessarily capture the geometric features of ligands bound to the channel cavity. The calculations herein give a detailed structural and energetic picture of blocker binding to Kv1.5 and this model should thus be useful for further ligand design efforts.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-101410 (URN)10.1529/biophysj.107.112045 (DOI)000252243200011 ()17905851 (PubMedID)
    Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2017-12-13Bibliographically approved
    5. Continuum solvation models in the linear interaction energy method
    Open this publication in new window or tab >>Continuum solvation models in the linear interaction energy method
    2006 In: Journal of Physical Chemistry B, ISSN 1520-6106, Vol. 110, no 24, p. 12034-12041Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-97212 (URN)
    Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved
  • 135.
    Andér, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Luzhkov, Victor B.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model2008In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 94, no 3, p. 820-831Article in journal (Refereed)
    Abstract [en]

    The binding of blockers to the human voltage-gated Kv1.5 potassium ion channel is investigated using a three-step procedure consisting of homology modeling, automated docking, and binding free energy calculations from molecular dynamics simulations, in combination with the linear interaction energy method. A reliable homology model of Kv1.5 is constructed using the recently published crystal structure of the Kv1.2 channel as a template. This model is expected to be significantly more accurate than earlier ones based on less similar templates. Using the three-dimensional homology model, a series of blockers with known affinities are docked into the cavity of the ion channel and their free energies of binding are calculated. The predicted binding free energies are in very good agreement with experimental data and the binding is predicted to be mainly achieved through nonpolar interactions, whereas the relatively small differences in the polar contribution determine the specificity. Apart from confirming the importance of residues V505, I508, V512, and V516 for ligand binding in the cavity, the results also show that A509 and P513 contribute significantly to the nonpolar binding interactions. Furthermore, we find that pharmacophore models based only on optimized free ligand conformations may not necessarily capture the geometric features of ligands bound to the channel cavity. The calculations herein give a detailed structural and energetic picture of blocker binding to Kv1.5 and this model should thus be useful for further ligand design efforts.

  • 136.
    Andér, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 15, p. 3483-3489Article in journal (Refereed)
    Abstract [en]

    The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination   reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic   structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is   no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.

  • 137.
    Ankarklev, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Inter and Intra-Assemblage Characterizations of Giardia intestinalis: from clinic to genome2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world.

    In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients.

    Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

    List of papers
    1. Dominance of Giardia assemblage B in León, Nicaragua.
    Open this publication in new window or tab >>Dominance of Giardia assemblage B in León, Nicaragua.
    Show others...
    2008 (English)In: Acta Tropica, ISSN 0001-706X, E-ISSN 1873-6254, Vol. 106, no 1, p. 44-53Article in journal (Refereed) Published
    Abstract [en]

    Giardiasis is a major problem in León, Nicaragua, yet despite this no data are available regarding the prevalence of different Giardia genotypes in this area. To address this question, a molecular analysis of Giardia isolates from humans and dogs living in the same area in León, Nicaragua was performed. Giardia isolates from 119 Nicaraguan patients and 8 dogs were successfully genotyped using single and/or nested beta-giardin PCR with subsequent restriction length fragment polymorphism (RFLP) analysis. The analyses of human samples yielded 94 (79%) assemblage B isolates and 25 (21%) assemblage A isolates. Only the non-human-associated assemblages C and D were found in the dog samples. Sixteen isolates with assemblage A pattern, 26 isolates with assemblage B pattern and all dog isolates were further characterized by sequencing the nested beta-giardin PCR product and by molecular analyses of the glutamate dehydrogenase (gdh) gene. Within the study area the assemblage A isolates were highly genetically homogenous, showing only sub-genotypes A2 (n=3) or A3 (n=13) at the beta-giardin locus and AII only at the gdh locus while assemblage B showed a high genetic polymorphism at both loci. Seven different sub-genotypes were identified within 13 of the sequenced assemblage B beta-giardin isolates. The remaining 13 sequenced assemblage B-isolates appeared to contain several different variants of the beta-giardin gene since the chromatograms displayed one to seven double peaks. The gdh sequences showed an even higher polymorphism since only 2 of 26 assemblage B isolates were without double peaks. Two mixed infections between assemblage A and B were found when the gdh gene was analyzed. Polymorphisms were also observed in the dog-associated assemblages C and D, but to a lesser extent than in assemblage B.

    Keywords
    Nicaragua, Giardia intestinalis, (-Giardin gene, glutamate dehydrogenase gene, genotyping, diarrhea
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-16512 (URN)10.1016/j.actatropica.2008.01.004 (DOI)000255487100007 ()18325480 (PubMedID)
    Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2017-12-08Bibliographically approved
    2. Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization
    Open this publication in new window or tab >>Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization
    2012 (English)In: PLoS Neglected Tropical Diseases, ISSN 1935-2735Article in journal (Other academic) Submitted
    Abstract [en]

    Background

    The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts world-wide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, little data is available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

    Methodology/Principal findings

    Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. No significant association was found in the studied population with regards to the presence of Giardia and gender, type of toilet, source of drinking-water or type of housing. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage A, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable and a significant association was found between Giardia assemblage B and H. pylori infection, OR=5.0 (1.9- 16).

    Conclusions/Significance

    This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that Giardia-infected children have a 3-fold higher risk of being infected by H. pylori. The data also suggests that assemblage B Giardia may be more closely associated with H. pylori infection.

    National Category
    Medical and Health Sciences Natural Sciences
    Research subject
    Molecular Medicine
    Identifiers
    urn:nbn:se:uu:diva-167046 (URN)
    Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2012-02-15Bibliographically approved
    3. Allelic sequence heterozygosity in single Giardia parasites
    Open this publication in new window or tab >>Allelic sequence heterozygosity in single Giardia parasites
    2012 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, article id 65Article in journal (Refereed) Published
    Abstract [en]

    Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

    Keywords
    Giardia, ASH, assemblage B, genotyping, single cell, micromanipulation
    National Category
    Medical and Health Sciences Natural Sciences
    Research subject
    Epidemiology; Biology with specialization in Microbiology
    Identifiers
    urn:nbn:se:uu:diva-167049 (URN)10.1186/1471-2180-12-65 (DOI)000308649900001 ()22554281 (PubMedID)
    Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2017-12-08Bibliographically approved
    4. Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.
    Open this publication in new window or tab >>Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.
    Show others...
    2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, p. 543-Article in journal (Refereed) Published
    Abstract [en]

    Background

    Giardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig.

    Results

    We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes.

    Conclusions

    Our results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.

    National Category
    Biological Sciences Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-133827 (URN)10.1186/1471-2164-11-543 (DOI)000283123400001 ()20929575 (PubMedID)
    Available from: 2010-11-19 Created: 2010-11-16 Last updated: 2017-12-12Bibliographically approved
    5. Genomic variation within Giardia intestinalis assemblage A isolates
    Open this publication in new window or tab >>Genomic variation within Giardia intestinalis assemblage A isolates
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic patients, to study the genetic diversity within assemblage A and to identify new assemblage A-specific genotyping targets.

    Results: Several biological differences between the assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 (AS98) and AII-2 (AS175)) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of an assemblage AI isolate (WB). Our analyses indicate that the divergence between AI and AII is approximately 1%, represented by ~100,000 single nucleotide polymorphisms (SNP). Moreover, SNPs are homogeneously distributed over the chromosomes with an enrichment in regions containing surface antigens and non-coding sequences. The level of allelic sequence heterozygosity (ASH) in the two AII isolates were found to be 0.25-0.35%, which is 25-30-fold higher than in the WB isolate. 37 proteinencoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolatespecific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 7 members, showed high sequence diversity between the assemblage A isolates and they could have important roles in hostparasite interactions. A subset of the variable genes was used to develop new genotyping methods for assemblage A isolates.

    Conclusions: Our results show that there is a significant genomic variation in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms. This identified putative virulence genes and generated a new assemblage A-specific genotyping approach.

    Keywords
    Giardia, comparative genomics, assemblage A, ASH, MLG
    National Category
    Medical and Health Sciences Natural Sciences
    Research subject
    Biology with specialization in Microbiology; Molecular Medicine; Infectious Diseases
    Identifiers
    urn:nbn:se:uu:diva-167052 (URN)
    Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2012-02-15
  • 138.
    Ankarklev, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization2012In: PLoS Neglected Tropical Diseases, ISSN 1935-2735Article in journal (Other academic)
    Abstract [en]

    Background

    The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts world-wide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, little data is available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

    Methodology/Principal findings

    Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. No significant association was found in the studied population with regards to the presence of Giardia and gender, type of toilet, source of drinking-water or type of housing. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage A, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable and a significant association was found between Giardia assemblage B and H. pylori infection, OR=5.0 (1.9- 16).

    Conclusions/Significance

    This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that Giardia-infected children have a 3-fold higher risk of being infected by H. pylori. The data also suggests that assemblage B Giardia may be more closely associated with H. pylori infection.

  • 139.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Franzen, Oscar
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden. KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Peirasmaki, Dimitra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jerlstrom-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lebbad, Marianne
    Publ Hlth Agcy Sweden, Dept Microbiol, SE-17182 Solna, Sweden..
    Andersson, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Bjorn
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;KISP, Sci Life Lab, S-17165 Solna, Sweden..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates2015In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, article id 697Article in journal (Refereed)
    Abstract [en]

    Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

  • 140.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hestvik, Elin
    Lebbad, Marianne
    Lindh, Johan
    Kaddu-Mulindwa, Deogratias H.
    Andersson, Jan O.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.
    Tylleskar, Thorkild
    Tumwine, James K.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children2012In: PLOS Neglected Tropical Diseases, ISSN 1935-2735, Vol. 6, no 8, p. e1780-Article in journal (Refereed)
    Abstract [en]

    Background: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

    Methodology/Principal Findings: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection.

    Conclusions/Significance: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.

  • 141.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jerlström-Hultqvist, Jon
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Ringqvist, Emma
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Troell, Karin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Behind the smile: cell biology and disease mechanisms of Giardia species2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 6, p. 413-422Article, review/survey (Refereed)
    Abstract [en]

    The eukaryotic intestinal parasite Giardia intestinalis was first described in 1681, when Antonie van Leeuwenhoek undertook a microscopic examination of his own diarrhoeal stool. Nowadays, although G. intestinalis is recognized as a major worldwide contributor to diarrhoeal disease in humans and other mammals, the disease mechanisms are still poorly understood. Owing to its reduced complexity and proposed early evolutionary divergence, G. intestinalis is used as a model eukaryotic system for studying many basic cellular processes. In this Review we discuss recent discoveries in the molecular cell biology and pathogenesis of G. intestinalis.

  • 142.
    Ankarklev, Johan
    et al.
    Stockholm Univ, Dept Mol Biosci, SE-10691 Stockholm, Sweden.
    Lebbad, Marianne
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Einarsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Franzen, Oscar
    Karolinska Inst, Integrated Cardio Metab Ctr, Novum, Box 285, SE-14157 Stockholm, Sweden.
    Ahola, Harri
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Troell, Karin
    Natl Vet Inst, Dept Microbiol, SE-75189 Uppsala, Sweden.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination2018In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 60, p. 7-16Article in journal (Refereed)
    Abstract [en]

    Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.

  • 143.
    Ankarklev, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lebbad, Marianne
    Allelic sequence heterozygosity in single Giardia parasites2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, article id 65Article in journal (Refereed)
    Abstract [en]

    Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

  • 144.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Baker, Louise
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Melbourne, Vic, Australia..
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun Div, Melbourne, Vic, Australia..
    Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 398Article in journal (Refereed)
    Abstract [en]

    Giardia duodenalis is an intestinal parasite that causes 200-300 million episodes of diarrhoea annually. Metronidazole (Mtz) is a front-line anti-giardial, but treatment failure is common and clinical resistance has been demonstrated. Mtz is thought to be activated within the parasite by oxidoreductase enzymes, and to kill by causing oxidative damage. In G. duodenalis, Mtz resistance involves active and passive mechanisms. Relatively low activity of iron-sulfur binding proteins, namely pyruvate: ferredoxin oxidoreductase (PFOR), ferredoxins, and nitroreductase-1, enable resistant cells to passively avoid Mtz activation. Additionally, low expression of oxygen-detoxification enzymes can allow passive (non-enzymatic) Mtz detoxification via futile redox cycling. In contrast, active resistance mechanisms include complete enzymatic detoxification of the pro-drug by nitroreductase-2 and enhanced repair of oxidized biomolecules via thioredoxin-dependent antioxidant enzymes. Molecular resistance mechanisms may be largely founded on reversible transcriptional changes, as some resistant lines revert to drug sensitivity during drug-free culture in vitro, or passage through the life cycle. To comprehensively characterize these changes, we undertook strand-specific RNA sequencing of three laboratory-derived Mtz-resistant lines, 106-2ID(10), 713-M3, and WB-M3, and compared transcription relative to their susceptible parents. Common up-regulated genes encoded variant-specific surface proteins (VSPs), a high cysteine membrane protein, calcium and zinc channels, a Mad-2 cell cycle regulator and a putative fatty acid a alpha-oxidase. Down-regulated genes included nitroreductase-1, putative chromate and quinone reductases, and numerous genes that act proximal to PFOR. Transcriptional changes in 106-2ID(10) diverged from those in 713-r and WB-r (r <= 0.2), which were more similar to each other (r = 0.47). In 106-2ID(10), a nonsense mutation in nitroreductase-1 transcripts could enhance passive resistance whereas increased transcription of nitroreductase-2, and a MATE transmembrane pump system, suggest active drug detoxification and efflux, respectively. By contrast, transcriptional changes in 713-M3 and WB-M3 indicated a higher oxidative stress load, attributed to Mtz- and oxygen-derived radicals, respectively. Quantitative comparisons of orthologous gene transcription between Mtz-resistant G. duodenalis and Trichomonas vaginalis, a closely related parasite, revealed changes in transcripts encoding peroxidases, heat shock proteins, and FMN-binding oxidoreductases, as prominent correlates of resistance. This work provides deep insight into Mtz-resistant G. duodenalis, and illuminates resistance-associated features across parasitic species.

  • 145.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Bio21 Mol Sci & Biotechnol Inst, Melbourne, Vic, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Emery, Samantha J.
    Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Svärd, Staffan G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Melbourne, Vic, Australia.;Walter & Eliza Hall Inst Med Res, Populat Hlth & Immun, Melbourne, Vic, Australia..
    Divergent Transcriptional Responses to Physiological and Xenobiotic Stress in Giardia duodenalis2016In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 60, no 10, p. 6034-6045Article in journal (Refereed)
    Abstract [en]

    Understanding how parasites respond to stress can help to identify essential biological processes. Giardia duodenalis is a parasitic protist that infects the human gastrointestinal tract and causes 200 to 300 million cases of diarrhea annually. Metronidazole, a major antigiardial drug, is thought to cause oxidative damage within the infective trophozoite form. However, treatment efficacy is suboptimal, due partly to metronidazole-resistant infections. To elucidate conserved and stress-specific responses, we calibrated sublethal metronidazole, hydrogen peroxide, and thermal stresses to exert approximately equal pressure on trophozoite growth and compared transcriptional responses after 24 h of exposure. We identified 252 genes that were differentially transcribed in response to all three stressors, including glycolytic and DNA repair enzymes, a mitogen-activated protein (MAP) kinase, high-cysteine membrane proteins, flavin adenine dinucleotide (FAD) synthetase, and histone modification enzymes. Transcriptional responses appeared to diverge according to physiological or xenobiotic stress. Downregulation of the antioxidant system and alpha-giardins was observed only under metronidazole-induced stress, whereas upregulation of GARP-like transcription factors and their subordinate genes was observed in response to hydrogen peroxide and thermal stressors. Limited evidence was found in support of stress-specific response elements upstream of differentially transcribed genes; however, antisense derepression and differential regulation of RNA interference machinery suggest multiple epigenetic mechanisms of transcriptional control.

  • 146.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Mol Sci & Biotechnol Inst Bio21, Parkville, Vic 3052, Australia..
    Baker, Louise
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Korhonen, Pasi K.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Young, Neil D.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Hall, Ross S.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Rojas, Cristian A. A.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3052, Australia..
    Time-Dependent Transcriptional Changes in Axenic Giardia duodenalis Trophozoites2015In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 9, no 12, article id e0004261Article in journal (Refereed)
    Abstract [en]

    Giardia duodenalis is the most common gastrointestinal protozoan parasite of humans and a significant contributor to the global burden of both diarrheal disease and post-infectious chronic disorders. Although G. duodenalis can be cultured axenically, significant gaps exist in our understanding of the molecular biology and metabolism of this pathogen. The present study employed RNA sequencing to characterize the mRNA transcriptome of G. duodenalis trophozoites in axenic culture, at log (48 h of growth), stationary (60 h), and declining (96 h) growth phases. Using similar to 400-times coverage of the transcriptome, we identified 754 differentially transcribed genes (DTGs), mainly representing two large DTG groups: 438 that were down-regulated in the declining phase relative to log and stationary phases, and 281 that were up-regulated. Differential transcription of prominent antioxidant and glycolytic enzymes implicated oxygen tension as a key factor influencing the transcriptional program of axenic trophozoites. Systematic bioinformatic characterization of numerous DTGs encoding hypothetical proteins of unknown function was achieved using structural homology searching. This powerful approach greatly informed the differential transcription analysis and revealed putative novel antioxidant-coding genes, and the presence of a nearcomplete two-component-like signaling system that may link cytosolic redox or metabolite sensing to the observed transcriptional changes. Motif searching applied to promoter regions of the two large DTG groups identified different putative transcription factor-binding motifs that may underpin global transcriptional regulation. This study provides new insights into the drivers and potential mediators of transcriptional variation in axenic G. duodenalis and provides context for static transcriptional studies.

  • 147.
    Ansell, Brendan R. E.
    et al.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    McConville, Malcolm J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Ma'ayeh, Showgy Y.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Dagley, Michael J.
    Univ Melbourne, Inst Bio21, Parkville, Vic 3010, Australia..
    Gasser, Robin B.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Svärd, Staffan G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Jex, Aaron R.
    Univ Melbourne, Fac Vet & Agr Sci, Parkville, Vic 3010, Australia..
    Drug resistance in Giardia duodenalis2015In: Biotechnology Advances, ISSN 0734-9750, E-ISSN 1873-1899, Vol. 33, no 6, p. 888-901Article, review/survey (Refereed)
    Abstract [en]

    Giardia duodenalis is a microaerophilic parasite of the human gastrointestinal tract and a major contributor to diarrheal and post-infectious chronic gastrointestinal disease world-wide. Treatment of G. duodenalis infection currently relies on a small number of drug classes. Nitroheterocyclics, in particular metronidazole, have represented the front line treatment for the last 40 years. Nitroheterocyclic-resistant G. duodenalis have been isolated from patients and created in vitro, prompting considerable research into the biomolecular mechanisms of resistance. These compounds are redox-active and are believed to damage proteins and DNA after being activated by oxidoreductase enzymes in metabolically active cells. In this review, we explore the molecular phenotypes of nitroheterocyclic-resistant G. duodenalis described to date in the context of the protisfs unusual glycolytic and antioxidant systems. We propose that resistance mechanisms are likely to extend well beyond currently described resistance-associated enzymes (i.e., pyruvate ferredoxin oxidoreductases and nitroreductases), to include NAD(P)H- and flavin-generating pathways, and possibly redox-sensitive epigenetic regulation. Mechanisms that allow G. duodenalis to tolerate oxidative stress may lead to resistance against both oxygen and nitroheterocyclics, with implications for clinical control. The present review highlights the potential for systems biology tools and advanced bioinformatics to further investigate the multifaceted mechanisms of nitroheterocyclic resistance in this important pathogen.

  • 148.
    Antoun, Ayman
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mechanism and Regulation of Initiation of Protein Synthesis in Eubacteria2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Initiation of protein synthesis in E.coli involves several steps, which lead to the formation of the first peptide bond. This process requires three initiation factors: IF1, IF2 and IF3. Using a novel technique of combined light scattering and stopped-flow, we elucidated the importance of IF2•GTP conformation for the recruitment of 50S to 30S pre-initiation complex. Moreover, GTP hydrolysis is essential for IF2 release and later binding of ternary complex. Interestingly, a switch in IF2 affinity to G-nucleotides is induced during 30S pre-initiation complexes formation.

    We found that IF1, previously with unknown functions in vitro, increases the rate of naked 70S dissociation by a factor 80 and acts as a fidelity factor in preventing 70S formation containing elongator tRNA instead of fMet-tRNAfMet. We showed that RRF/EFG/IF3 split both naked and post-termination complexes while IF1/IF3 split only naked ribosomes. The mechanisms of action of RRF/ EFG, the order of their binding to 70S, as well as, the three different conformation of EF-G on the ribosomes are emphasized. Interestingly, 70S formation rate is dependent on the concentration of IF3 and not linear with 50S subunits concentration. We demonstrated that the rate-limiting step in 70S formation is IF3 dissociation from 30S complexes.

    The interplay between initiation factors in the rate and accuracy of protein synthesis was thoroughly studied. Using fMet-tRNAfMet (initiator tRNA), Met-tRNAfMet (non-formylated initiator tRNA) and Phe-tRNAPhe (elongator tRNA), we showed that the major player in the accuracy is IF2 through recognizing the formyl group on fMet-tRNAfMet, while IF3 acts by increasing both the on- and off-rate of tRNA from 30S pre-initiation complexes.

    Collectively, these novel results describe a comprehensive model of initiation of protein synthesis. In this model, initiation factors increase the rate of fMet-tRNAfMet binding to 30S subunits, subsequently; the stabilization of fMet-tRNAfMet by IF2 increases the rate of IF3 dissociation. Later, IF2 in GTP conformation allows 50S docking to 30S pre-initiation complex free of IF3 followed by GTP hydrolysis allowing IF2 release for ternary complex to bind and start elongation of protein synthesis.

    List of papers
    1. The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis
    Open this publication in new window or tab >>The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis
    Show others...
    2003 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 22, no 20, p. 5593-5601Article in journal (Refereed) Published
    Abstract [en]

    The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

    Keywords
    G protein, IF2, initiation of translation, protein synthesis, ribosome
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-93386 (URN)10.1093/emboj/cdg525 (DOI)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2017-12-14Bibliographically approved
    2. Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering
    Open this publication in new window or tab >>Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering
    2004 In: Biological procedure online, Vol. 6, no 1, p. 35-54Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-93387 (URN)
    Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved
    3. A time-resolved investigation of ribosomal subunit association
    Open this publication in new window or tab >>A time-resolved investigation of ribosomal subunit association
    Show others...
    2005 In: Journal of Molecular Biology, Vol. 346, no 5, p. 1243-1258Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-93388 (URN)
    Available from: 2005-09-01 Created: 2005-09-01Bibliographically approved
    4. Interplay of initiation factors and initiator tRNA in initiation of eubacterial protein synthesis
    Open this publication in new window or tab >>Interplay of initiation factors and initiator tRNA in initiation of eubacterial protein synthesis
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93389 (URN)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
    5. Accuracy of initiator tRNA selection in initiation of eubacterial protein synthesis
    Open this publication in new window or tab >>Accuracy of initiator tRNA selection in initiation of eubacterial protein synthesis
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93390 (URN)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
    6. Characterization of the GTP Binding Properties of Initiation Factor 2
    Open this publication in new window or tab >>Characterization of the GTP Binding Properties of Initiation Factor 2
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93391 (URN)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
    7. Why is initiation factor IF1 essential for E.Coli?
    Open this publication in new window or tab >>Why is initiation factor IF1 essential for E.Coli?
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93392 (URN)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
    8. Ribosomal recycling by the interplay of RRF and three conformations of EFG
    Open this publication in new window or tab >>Ribosomal recycling by the interplay of RRF and three conformations of EFG
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-93393 (URN)
    Available from: 2005-09-01 Created: 2005-09-01 Last updated: 2010-01-13Bibliographically approved
  • 149.
    Antoun, Ayman
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    The roles of initiation factor IF2 and GTP in initiation of protein synthesis2003Licentiate thesis, monograph (Other scientific)
  • 150.
    Antoun, Ayman
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    Ehrenberg, Måns
    Characterization of the GTP Binding Properties of Initiation Factor 2Manuscript (Other academic)
1234567 101 - 150 of 3209
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