umu.sePublications
Change search
Refine search result
2345678 201 - 250 of 802
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 201. Falahati, Hanieh
    et al.
    Pazhang, Mohammad
    Zareian, Shekufeh
    Ghaemi, Nasser
    Rofougaran, Reza
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rezaie, Alireza R
    Khajeh, Khosro
    Transmitting the allosteric signal in methylglyoxal synthase2013In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 26, no 7, p. 445-452Article in journal (Refereed)
    Abstract [en]

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

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

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

  • 203. Felten, Judith
    et al.
    Hall, Hardy
    Jaumot, Joaquim
    Tauler, Roma
    de Juan, Anna
    Gorzsas, Andras
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vibrational spectroscopic image analysis of biological material using multivariate curve resolution-alternating least squares (MCR-ALS)2015In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, no 2, p. 217-240Article in journal (Refereed)
    Abstract [en]

    Raman and Fourier transform IR (FTIR) microspectroscopic images of biological material (tissue sections) contain detailed information about their chemical composition. The challenge lies in identifying changes in chemical composition, as well as locating and assigning these changes to different conditions (pathology, anatomy, environmental or genetic factors). Multivariate data analysis techniques are ideal for decrypting such information from the data. This protocol provides a user-friendly pipeline and graphical user interface (GUI) for data pre-processing and unmixing of pixel spectra into their contributing pure components by multivariate curve resolution-alternating least squares (MCR-ALS) analysis. The analysis considers the full spectral profile in order to identify the chemical compounds and to visualize their distribution across the sample to categorize chemically distinct areas. Results are rapidly achieved (usually <30-60 min per image), and they are easy to interpret and evaluate both in terms of chemistry and biology, making the method generally more powerful than principal component analysis (PCA) or heat maps of single-band intensities. In addition, chemical and biological evaluation of the results by means of reference matching and segmentation maps (based on k-means clustering) is possible.

  • 204.
    Figueiredo, Margarida L. A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kim, Maria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå University, SE-90187 Umeå, Sweden.
    Allgardsson, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Computational Life Science Cluster (CLiC), Umeå UniversityUmeå, Sweden; Division of CBRN Defence and Security, FOI, Swedish Defence Research Agency, Sweden.
    Larsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Non-coding roX RNAs prevent the binding of the MSL-complex to heterochromatic regions2014In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, p. e1004865-Article in journal (Refereed)
    Abstract [en]

    Long non-coding RNAs contribute to dosage compensation in both mammals and Drosophila by inducing changes in the chromatin structure of the X-chromosome. In Drosophila melanogaster, roX1 and roX2 are long non-coding RNAs that together with proteins form the male-specific lethal (MSL) complex, which coats the entire male X-chromosome and mediates dosage compensation by increasing its transcriptional output. Studies on polytene chromosomes have demonstrated that when both roX1 and roX2 are absent, the MSL-complex becomes less abundant on the male X-chromosome and is relocated to the chromocenter and the 4thchromosome. Here we address the role of roX RNAs in MSL-complex targeting and the evolution of dosage compensation in Drosophila. We performed ChIP-seq experiments which showed that MSL-complex recruitment to high affinity sites (HAS) on the X-chromosome is independent of roX and that the HAS sequence motif is conserved in D. simulans. Additionally, a complete and enzymatically active MSL-complex is recruited to six specific genes on the 4thchromosome. Interestingly, our sequence analysis showed that in the absence of roX RNAs, the MSL-complex has an affinity for regions enriched in Hoppel transposable elements and repeats in general. We hypothesize that roX mutants reveal the ancient targeting of the MSL-complex and propose that the role of roX RNAs is to prevent the binding of the MSL-complex to heterochromatin.

  • 205.
    Fijolek, Artur
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Salvage and de novo synthesis of nucleotides in Trypanosoma brucei and mammalian cells2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    All living cells are dependent on nucleic acids for their survival. The genetic information stored in DNA is translated into functional proteins via a messenger molecule, the ribonucleic acid (RNA). Since DNA and RNA can be considered as polymers of nucleotides (NTPs), balanced pools of NTPs are crucial to nucleic acid synthesis and repair. The de novo reduction of ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs), the precursors for DNA synthesis, is catalyzed by the enzyme ribonucleotide reductase (RNR). In cycling cells the dominant form of mammalian RNR consists of two proteins called R1 and R2. A proteasome-mediated degradation completely deprives postmitotic cells of R2 protein. The nonproliferating cells use instead a p53 inducible small RNR subunit, called p53R2 to synthesize dNTPs for mitochondrial DNA replication and DNA repair. To address the ongoing controversy regarding the localization and subsequently function and regulation of RNR subunits, the subcellular localization of all the mammalian RNR subunits during the cell cycle and after DNA damage was followed as a part of this thesis. Irrespective of the employed methodology, only a cytosolic localization could be observed leading to a conclusion that the dNTPs are synthesized in the cytosol and transported into the nucleus or mitochondria for DNA synthesis and repair. Thus, our data do not support the suggestion that nuclear translocation is a new additional mechanism regulating ribonucleotide reduction in mammalian cells.

    In an attempt to find a cure for African sleeping sickness, a lethal disease caused by a human pathogen, Trypanosoma brucei, nucleotide metabolism of the parasite was studied. The trypanosomes exhibit strikingly low CTP pools compared with mammalian cells and they also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. Following expression, purification and kinetic studies of the recombinant T. brucei CTP synthetase it was found that the enzyme has a higher Km value for UTP than the mammalian CTP synthetase. In combination with a lower UTP pool the high Km may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase was irreversibly inhibited by the glutamine analog acivicin, a drug extensively tested as an antitumor agent. Daily injections of acivicin to trypanosome-infected mice were sufficient to suppress the parasite infections. The drug was shown to be trypanocidal when added to cultured bloodstream T. brucei for four days at 1 uM concentration. Therefore, acivicin may qualify as a drug with “desirable” properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi. Trypanosomes lack de novo purine biosynthesis and are therefore dependent on exogenous purines such as adenosine that is taken up from the blood by high-affinity transporters. We found that besides the cleavage-dependent pathway, where adenosine is converted to adenine by inosine-adenosine-guanosine-nucleoside hydrolase, T. brucei can also salvage adenosine by adenosine kinase (AK). The efficient adenosine transport combined with a high-affinity AK yields a strong salvage system in T. brucei, but on the other hand makes the parasites highly sensitive to adenosine analogs such as adenine arabinoside (Ara-A). The cleavage-resistant Ara-A was shown to be readily taken up by the parasites and phosphorylated by the TbAK-dependent pathway, inhibiting trypanosome proliferation and survival by incorporation into nucleic acids and by affecting nucleotide levels in the parasite.

  • 206.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Curth, Ute
    Pingoud, Alfred
    Wende, Wolfgang
    Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 2, p. 847-860Article in journal (Refereed)
    Abstract [en]

    Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.

  • 207.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany.
    Chylinski, Krzysztof
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Deptartment of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Austria.
    Makarova, Kira S.
    Lécrivain, Anne-Laure
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bzdrenga, Janek
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Koonin, Eugene V.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig, Germany ; Hannover Medical School, Hannover, Germany .
    Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems2014In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 42, no 4, p. 2577-2590Article in journal (Refereed)
    Abstract [en]

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA: crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA: Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.

  • 208.
    Fonfara, Ines
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    Richter, Hagen
    Bratovic, Majda
    Le Rhun, Anaïs
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Helmholtz Centre for Infection Research, Department of Regulation in Infection Biology, Braunschweig 38124, Germany; 3Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin 10117, Germany.
    The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA2016In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 532, no 7600, p. 517-520Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II and III utilize a set of distinct CRISPR-associated ( Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA)(2-5) and the mature crRNAs then guide a complex of Cas proteins ( Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA(6-12). In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA15. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN- 3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang(16). The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V- A constitutes the most minimalistic of the CRISPR- Cas systems so far described.

  • 209.
    Forsgren, Nina
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Lamont, Richard J
    Persson, Karina
    Umeå University, Faculty of Medicine, Department of Odontology, Cariology.
    Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB2009In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 9, p. 1896-1905Article in journal (Refereed)
    Abstract [en]

    The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

  • 210.
    Forshell, Linus Plym
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Li, Yongmei
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Plym Forshell, Tacha Zi
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Rudelius, Martina
    Department of Pathology, Technical University of Munich, Munich, Germany.
    Nilsson, Lisa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Keller, Ulrich
    Department of Medicine, Technical University of Munich, Munich, Germany.
    Nilsson, Jonas
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The direct Myc target Pim3 cooperates with other Pim kinases in supporting viability of Myc-induced B-cell lymphomas2011In: Oncotarget, ISSN 1949-2553, Vol. 2, no 6, p. 448-460Article in journal (Refereed)
    Abstract [en]

    The Pim kinases are weak oncogenes. However, when co-expressed with a strong oncogene, such as c-Myc, Pim kinases potentiate the oncogenic effect resulting in an acceleration of tumorigenesis. In this study we show that the least studied Pim kinase, Pim-3, is encoded by a gene directly regulated by c-Myc via binding to one of the conserved E-boxes within the Pim3 gene. Accordingly, lymphomas arising in Myc-transgenic mice and Burkitt lymphoma cell lines exhibit elevated levels of Pim-3. Interestingly, inhibition of Pim kinases by a novel pan-Pim kinase inhibitor, Pimi, in Myc-induced lymphoma results in cell death that appears independent of caspases. The data indicate that Pim kinase inhibition could be a viable treatment strategy in certain human lymphomas that rely on Pim-3 kinase expression.

  • 211.
    Forslund, Anna-Lena
    Umeå University, Faculty of Medicine, Molecular Biology.
    Identification of new virulence factors in Francisella tularensis2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq.

    The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain.

    In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.

  • 212.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Salomonsson, Emelie
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Goloviov, Igor
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
    Kuoppa, Kerstin
    FOI, Umeå (Swedish Defence Research Agency).
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI, Umeå (Swedish Defence Research Agency).
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Bacteriology.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Biology.
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensisManuscript (Other (popular science, discussion, etc.))
    Abstract [en]

    Background: All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.

    Results: In a previous study, we were able to show that one pilin gene, pilA, was essential for virulence of a type B strain in a mouse infection model. In this work we have examined the role of several pilin genes in the virulence of the pathogenic type A strain SCHU S4. pilA, pilC, pilQ, and pilT were mutated by in-frame deletion mutagenesis. Interestingly, when mice were infected with a mixture of each mutant strain and the wild-type strain, the pilA, pilC and pilQ mutants were out-competed, while the pilT mutant was equally competitive as the wild-type.

    Conclusions: This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

  • 213.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The pathogenic Yersiniae: advances in the understanding of physiology and virulence2013In: Frontiers in Cellular and Infection Microbiology, ISSN 2235-2988, Vol. 3, no 51, p. 2p. 1-2Article, review/survey (Refereed)
  • 214.
    Francis, Matthew S
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Amer, Ayad A A
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Milton, Debra
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Biological and Environmental Sciences, Troy University, Troy, AL, USA.
    Costa, Tiago R D
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Institute of Structural and Molecular Biology, University College London and Birkbeck, London, UK.
    Site-directed mutagenesis and its application in studying the interactions of T3S components2017In: Type 3 secretion systems: methods and protocols / [ed] Matthew L. Nilles and Danielle L. Jessen Condry, Humana Press, 2017, p. 11-31Chapter in book (Refereed)
    Abstract [en]

    Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections—unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

  • 215.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Vidal-Quadras, Maite
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Henriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Santarella-Mellwig, Rachel
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF12015In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 22, p. 4183-4195Article in journal (Refereed)
    Abstract [en]

    Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase activating protein (GAP) GRAF1, facilitate rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, distinct from clathrin coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane resulting in a corresponding decrease in Cdc42 microdomain association. However, Cdc42 captured in its active state was, via a GAP domain mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers affected in their endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

  • 216.
    Francis, Monika K.
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Krupp, Nikolai
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Behrmann, Elmar
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    GRAF1 sculpts membrane through a regulated oligomerisation reactionManuscript (preprint) (Other academic)
  • 217.
    Friberg, Josefin
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    The control of growth and metabolism in Caenorhabditis elegans2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The control of growth is a poorly understood aspect of animal development. This thesis focuses on body size regulation in Caenorhabditis elegans, and in particular, how worms grow to a certain size.

    In C. elegans, a key regulator of size is the TGFβ homologue DBL-1. Mutations that deplete the worm of DBL-1 result in a small body size, whereas overexpression of the gene renders long animals. The small mutants have the same number of cells as wild type suggesting that some or all cells are smaller. DBL-1 activates a TGFβ receptor leading to the nuclear localization of three Smad proteins which then initiate a transcriptional program for size control whose targets are mainly unknown. In order to learn more about how body size in C. elegans is regulated, we set up EMS mutagenesis screens to identify new loci that caused a long phenotype. A subset of the genes we have identified might function in the TGFβ signaling pathway regulating growth while others likely function in parallel pathways.

    One gene that we found in this screen, lon-3, encodes a cuticle collagen that genetically lies downstream of the DBL-1 TGFβ signaling pathway. Interestingly, loss of function mutations in lon-3 result in a Lon phenotype, whereas increasing the amount of LON-3 protein cause the worms to be dumpy, i.e. shorter, but slightly fatter than wild type. LON-3 is expressed in the hypodermis, the tissue from which the cuticle is synthesized and in which TGFβ signaling, regulating body size, has its focus. This study and previous work have shown that DBL-1 may affect body volume via effects on hypodermal nuclear ploidy, however this is unaffected in lon-3 mutants. Consistent with this finding, the volume of lon-3 mutant worms is not different from wild type. Taken together, our results suggest that another mechanism, by which TGFβ signaling can regulate body length, is by altering the shape of the cuticle via its effect on lon-3 and possibly other cuticle collagens.

    Studies in worms, flies and mice show that body size and nutrient allocation are closely connected. p70 S6-kinase (S6K) is a known regulator of cell and body size that also plays a role in metabolism. In mice and flies S6K mutants are much smaller than wild type. Our work on the worm homolog, rsks-1, shows that in worms as well, this gene is important for growth regulation and cell size. However, this effect seems to be at least in part independent of DBL-1 TGFβ signaling. Furthermore, rsks-1mutants have a 50 % increase in the amount of stored fat. Fatty acid metabolism has been shown to play an important role in environmental adaptation, especially in regards to temperature changes. Consistent with this idea, rsks-1 mutants appear to have difficulties in adjusting to such changes, reflected in a much-decreased fecundity at 15 and 25 °C compared to their cultivation temperature (20 °C).

    Within the nervous system the gene is specifically expressed in a subset of the chemosensory neurons that, when nutrients are abundant, secrete signals that promote growth. Intriguingly, this expression seems to be negatively regulated by insulin- like signaling, in contrast to the positive regulation of S6K by insulin in Drosophila and mice. Taken together we show that rsks-1 is an important regulator of growth and fat metabolism in Caenorhabditis elegans.

  • 218.
    Frithz-Lindsten, Elisabet
    et al.
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Holmström, Anna
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Jacobsson, Lars
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Soltani, Mehnam
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Olsson, Jan
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Department of Microbiology, Defence Research Establishment, S-901 82, Umeå, Sweden.
    Functional conservation of the effector protein translocators PopB/YopB and PopD/YopD of Pseudomonas aeruginosa and Yersinia pseudotuberculosis.1998In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 29, no 5, p. 1155-1165Article in journal (Refereed)
    Abstract [en]

    Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

  • 219. Frykholm, Karolin
    et al.
    Berntsson, Ronnie Per-Arne
    Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden.
    Claesson, Magnus
    de Battice, Laura
    Odegrip, Richard
    Stenmark, Pål
    Westerlund, Fredrik
    DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 15, p. 7219-7227Article in journal (Refereed)
    Abstract [en]

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and W Phi affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and W Phi Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.

  • 220. Gahan, CS
    et al.
    Sundkvist, JE
    Dopson, Mark
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Sandström, A
    Effect of chloride on ferrous iron oxidation by a leptospirillum ferriphilum-dominated chemostat culture2010In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 106, no 3, p. 422-431Article in journal (Refereed)
    Abstract [en]

    Biomining is the use of microorganisms to catalyze metal extraction from sulfide ores. However, the available water in some biomining environments has high chloride concentrations and therefore, chloride toxicity to ferrous oxidizing microorganisms has been investigated. Batch biooxidation of Fe2+ by a Leptospirillum ferriphilum dominated culture was completely inhibited by 12gL(-1) chloride. In addition, the effects of chloride on oxidation kinetics in a Fe2+ limited chemostat were studied. Results from the chemostat modeling suggest that the chloride toxicity was attributed to affects on the Fe2+ oxidation system, pH homeostasis, and lowering of the proton motive force. Modeling showed a decrease in the maximum specific growth rate (mu(max)) and an increase in the substrate constant (K-s) with increasing chloride concentrations, indicating an effect on the Fe2+ oxidation system. The model proposes a lowered maintenance activity when the media was fed with 2-3 g L-1 chloride with a concomitant drastic decrease in the true yield (Y-true). This model helps to understand the influence of chloride on Fe2+ biooxidation kinetics. Biotechnol. Bioeng. 2010;106: 422-431. (C) 2010 Wiley Periodicals, Inc.

  • 221.
    Gallampois, Christine M. J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Effect-Directed Analysis, UFZ—Helmholtz Centre for Environmental Research, Leipzig, Germany.
    Schymanski, Emma L.
    Bataineh, Mahmoud
    Buchinger, Sebastian
    Krauss, Martin
    Reifferscheid, Georg
    Brack, Werner
    Integrated biological-chemical approach for the isolation and selection of polyaromatic mutagens in surface waters2013In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 405, no 28, p. 9101-9112Article in journal (Refereed)
    Abstract [en]

    Many environmental mutagens, including polyaromatic compounds are present in surface waters, often in complex mixtures and at low concentrations. The present study provides and applies a novel, integrated approach to isolate polyaromatic mutagens in river water using a sample from the River Elbe. The sample was taken downstream of industrial discharges using blue rayon (BR) as a passive sampler that selectively adsorbs polyaromatic compounds and was subjected to effect-directed fractionation in order to characterise the compounds causing the detected effect(s). The procedure relies on three complementary fractionation steps, the Ames fluctuation assay with strains TA98, YG1024 and YG1041 with and without S9 activation and analytical screening. Several mutagenic fractions were isolated by combining mutagenicity testing with fractionation. The enhanced mutagenicity in the nitroreductase and/or O-acetyltransferase overexpressing strains YG1024 and YG1041 strains suggested amino- and/or nitro-compounds causing mutagenicity in several fractions. Analytical screening of mutagenic fractions with LC-HRMS/MS provided a list of molecular formulas typically containing one to ten nitrogen and at least two oxygen atoms supporting the presence of amino and nitro-compounds in the mutagenic fractions.

  • 222.
    Ganai, Rais Ahmad
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Structural and biochemical basis for the high fidelity and processivity of DNA polymerase ε2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    DNA polymerase epsilon (Pol ε) is a multi-subunit B-family DNA polymerase that is involved in leading strand DNA replication in eukaryotes. DNA Pol ε in yeast consists of four subunits, Pol2, Dpb2, Dpb3, and Dpb4. Pol2 is the catalytic subunit and Dpb2, Dpb3, and Dpb4 are the accessory subunits. Pol2 can be further divided into an N-terminal catalytic core (Pol2core) containing both the polymerase and exonuclease active sites and a C-terminus domain. We determined the X-ray crystal structure of Pol2core at 2.2 Å bound to DNA and with an incoming dATP. Pol ε has typical fingers, palm, thumb, exonuclease, and N-terminal domains in common with all other B-family DNA polymerases. However, we also identified a seemingly novel domain we named the P-domain that only appears to be present in Pol ε. This domain partially encircles the nascent duplex DNA as it leaves the active site and contributes to the high intrinsic processivity of Pol ε.

    To ask if the crystal structure of Pol2core can serve as a model for catalysis by Pol ε, we investigated how the C-terminus of Pol2 and the accessory subunits of Pol ε influence the enzymatic mechanism by which Pol ε builds new DNA efficiently and with high fidelity. Pre-steady state kinetics revealed that the exonuclease and polymerization rates were comparable between Pol2core and Pol ε. However, a global fit of the data over five nucleotide-incorporation events revealed that Pol ε is slightly more processive than Pol2 core. The largest differences were observed when measuring the time for loading the polymerase onto a 3' primer-terminus and the subsequent incorporation of one nucleotide. We found that Pol ε needed less than a second to incorporate the first nucleotide, but it took several seconds for Pol2core to incorporate similar amounts of the first nucleotide.

    B-family polymerases have evolved an extended β-hairpin loop that is important for switching the primer terminus between the polymerase and exonuclease active sites. The high-resolution structure of Pol2core revealed that Pol ε does not possess an extended β-hairpin loop. Here, we show that Pol ε can processively transfer a mismatched 3' primer-terminus between the polymerase and exonuclease active sites despite the absence of a β-hairpin loop. Additionally we have characterized a series of amino acid substitutions in Pol ε that lead to altered partitioning of the 3'primer-terminus between the two active sites.

    In a final set of experiments, we investigated the ability of Pol ε to displace the downstream double-stranded DNA while carrying out DNA synthesis. Pol ε displaced only one base pair when encountering double-stranded DNA after filling a gap or a nick. However, exonuclease deficient Pol ε carries out robust strand displacement synthesis and can reach the end of the templates tested here. Similarly, an abasic site or a ribonucleotide on the 5'-end of the downstream primer was efficiently displaced but still only by one nucleotide. However, a flap on the 5'-end of the blocking primer resembling a D-loop inhibited Pol ε before it could reach the double-stranded junction. Our results are in agreement with the possible involvement of Pol ε in short-patch base excision repair and ribonucleotide excision repair but not in D-loop extension or long-patch base excision repair.

  • 223.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bylund, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Switching between polymerase and exonuclease sites in DNA polymerase ε2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 2, p. 932-942Article in journal (Refereed)
    Abstract [en]

    The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases.

  • 224.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NY 10016, USA.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    DNA Replication - A Matter of Fidelity2016In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 62, no 5, p. 745-755Article, review/survey (Refereed)
    Abstract [en]

    The fidelity of DNA replication is determined by many factors, here simplified as the contribution of the DNA polymerase (nucleotide selectivity and proofreading), mismatch repair, a balanced supply of nucleotides, and the condition of the DNA template (both in terms of sequence context and the presence of DNA lesions). This review discusses the contribution and interplay between these factors to the overall fidelity of DNA replication.

  • 225.
    Ganai, Rais Ahmad
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Osterman, Pia
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Erik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Yeast DNA Polymerase epsilon Catalytic Core and Holoenzyme Have Comparable Catalytic Rates2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3825-3835Article in journal (Refereed)
    Abstract [en]

    The holoenzyme of yeast DNApolymerase ε (Pol ε) consists of four subunits– Pol2, Dpb2, Dpb3, and Dpb4. A proteasesensitivesite results in a N-terminalproteolytic fragment of Pol2, called Pol2core,that consists of the catalytic core of Pol ε andretains both polymerase and exonucleaseactivities. Pre-steady-state kinetics showedthat the exonuclease rates on single-stranded,double-stranded, and mismatched DNA werecomparable between Pol ε and Pol2core. Singleturnover pre-steady-state kinetics alsoshowed that the kpol of Pol ε and Pol2core werecomparable when pre-loading the polymeraseonto the primer-template before adding Mg2+and dTTP. However, a global fit of the dataover six sequential nucleotide incorporationsrevealed that the overall polymerization rateand processivity was higher for Pol ε than forPol2core. The largest difference was observedwhen challenged for the formation of aternary complex and incorporation of thefirst nucleotide. Pol ε needed less than asecond to incorporate a nucleotide, butseveral seconds passed before Pol2coreincorporated detectable levels of the firstnucleotide. We conclude that the accessorysubunits and the C-terminus of Pol2 do notinfluence the catalytic rate of Pol ε butfacilitate the loading and incorporation of thefirst nucleotide by Pol ε.

  • 226.
    Garcia-Lorenzo, Maribel
    Umeå University, Faculty of Science and Technology, Chemistry.
    The Role of Proteases in Plant Development2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution.

    Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization.

    The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important.

    The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro. Arabidopsis knockout mutants confirmed the function of FtsH6 in vivo.

  • 227. Ge, Changrong
    et al.
    Georgiev, Alexander
    Öhman, Anders
    Wieslander, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Kelly, Amelie A.
    Tryptophan Residues Promote Membrane Association for a Plant Lipid Glycosyltransferase Involved in Phosphate Stress2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 8, p. 6669-6684Article in journal (Refereed)
    Abstract [en]

    Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

  • 228.
    Gharibyan, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Raveh, Dina
    Department of Life Sciences, Ben Gurion University of the Negev, Israel.
    Morozova-Roche, Ludmilla
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    S100A8/A9 amyloidosis in the ageing prostate: relating ex vivo and in vitro studies2012In: Amyloid Proteins: Methods and Protocols / [ed] Einar M. Sigurdsson, Miguel Calero, María Gasset, Springer Science+Business Media B.V., 2012, Vol. 849, p. 387-401Chapter in book (Refereed)
    Abstract [en]

    The family of S100 proteins encompasses more than 20 members characterized by remarkable conformational and functional diversity. S100 proteins act as central regulators of various cellular processes, including cell survival, proliferation, differentiation, and motility. Many S100 proteins are implicated in various types of cancer as well as neurodegenerative, inflammatory, and autoimmune diseases. Recently, we have found that S100A8⁄A9 proteins are involved in amyloidogenic process in the ageing prostate, contributing to the formation of calcified corpora amylacea (CA) inclusions, which commonly accompany age-dependent prostate tissue remodelling and cancer. Amyloid formation by S100A8/A9 proteins can also be modelled in vitro. Amyloid assembly of S100A8/A9 proteins into oligomeric and fibrillar complexes is modulated by metal ions such as calcium and zinc. Here, we provide insights into the extraction procedures and review the common structural features of ex vivo and in vitro S100A8/A9 amyloids, showing that they share the same generic origin.

  • 229.
    Gharizadeh, Baback
    et al.
    Stanford Genome Technology Center, Stanford University, Palo Alto, USA.
    Eriksson, Jonas
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nourizad, Nader
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nordström, Tommy
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Nyrén, Pål
    Department of Biotechnology, Stockholm Center for Physics, Astronomy and Biotechnology, Royal Institute of Technology, Stockholm.
    Improvements in Pyrosequencing technology by employing Sequenase polymerase2004In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 330, no 2, p. 272-280Article in journal (Refereed)
    Abstract [en]

    Pyrosequencing is a DNA sequencing technique based on the bioluminometric detection of inorganic pyrophosphate, which is released when nucleotides are incorporated into a target DNA. Since the technique is based on an enzymatic cascade, the choice of enzymes is a critical factor for efficient performance of the sequencing reaction. In this study we have analyzed the performance of an alternative DNA polymerase, Sequenase, on the sequencing performance of the Pyrosequencing technology. Compared to the Klenow fragment of DNA polymerase I, Sequenase could read through homopolymeric regions with more than five T bases. In addition, Sequenase reduces remarkably interference from primer-dimers and loop structures that give rise to false sequence signals. By using Sequenase, synchronized extensions and longer reads can be obtained on challenging templates, thereby opening new avenues for applications of Pyrosequencing technology.

  • 230. Ghssein, Ghassan
    et al.
    Brutesco, Catherine
    Ouerdane, Laurent
    Fojcik, Clementine
    Izaute, Amelie
    Wang, Shuanglong
    Hajjar, Christine
    Lobinski, Ryszard
    Lemaire, David
    Richaud, Pierre
    Voulhoux, Rome
    Espaillat, Akbar
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pignol, David
    Borezee-Durant, Elise
    Arnoux, Pascal
    Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 352, no 6289, p. 1105-1109Article in journal (Refereed)
    Abstract [en]

    Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.

  • 231.
    Girma, Misgina Belachew
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kifle, Demeke
    Univ Addis Ababa, Dept Biol, Addis Ababa, Ethiopia.
    Jebessa, Habte
    Univ Addis Ababa, Dept Biol, Addis Ababa, Ethiopia.
    Deep underwater seismic explosion experiments and their possible ecological impact: The case of Lake Arenguade-Central Ethiopian highlands2012In: Limnologica, ISSN 0075-9511, E-ISSN 1873-5851, Vol. 42, no 3, p. 212-219Article in journal (Refereed)
    Abstract [en]

    The study was conducted in Lake Arenguade (Lake Haro Hadho) from 2008 to 2009 and results were compared with previous studies conducted by different authors since the 1960s. The study included the chemistry and chlorophyll-a biomass in micrograms per liter (mu g L-1). Results showed that chlorophylla biomass dramatically decreased since the 1960s. Previous studies indicated that the phytoplankton community of Lake Arenguade was dominated by a single cyanobacterium species, Arthrospira fusiforrnis (Voronichin) Komarek et Lund (syn. Spirulina fusiformis Voronichin) while the present study showed co-dominance of the lake's phytoplankton by another cyanobacterium species, Anabaenopsis elenkinii Miller. The trend shows that A. fusiformis is on the verge of disappearance from Lake Arenguade. While other factors can be responsible for such a change, the contribution of underwater seismological detonation experiments carried out repeatedly cannot be ruled out. Based on the results, recommendations were forwarded for possible full-fledged environmental impact assessment of explosion experiments in Lake Arenguade; and other lakes in which similar explosion experiments were carried out. (C) 2012 Elsevier GmbH. All rights reserved.

  • 232.
    Good, James A. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Christopher
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hansen, Sabine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wall, Jessica
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Krishnan, Syam
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Begum, Afshan
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Niemiec, Moritz Sebastian
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sauer, Uwe H.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sauer–Eriksson, A. Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA2016In: Cell chemical biology, ISSN 2451-9448, Vol. 23, no 3, p. 404-414Article in journal (Refereed)
    Abstract [en]

    The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

  • 233.
    Good, James A. D.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Silver, Jim
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nunez-Otero, Carlos
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bahnan, Wael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Salin, Olli
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Engström, Patrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Svensson, Richard
    Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden; The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden, Uppsala University, SE-751 23 Uppsala, Sweden.
    Artursson, Per
    Department of Pharmacy, Uppsala University, SE-751 23 Uppsala, Sweden; The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden, Uppsala University, SE-751 23 Uppsala, Sweden.
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thiazolino 2-Pyridone Amide Inhibitors of Chlamydia trachomatis Infectivity2016In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 5, p. 2094-2108Article in journal (Refereed)
    Abstract [en]

    The bacterial pathogen Chlamydia trachomatis is a global health burden currently treated with broad-spectrum antibiotics which disrupt commensal bacteria. We recently identified a compound through phenotypic screening that blocked infectivity of this intracellular pathogen without host cell toxicity (compound 1, KSK 120). Herein, we present the optimization of 1 to a class of thiazolino 2-pyridone amides that are highly efficacious (EC50 <= 100 nM) in attenuating infectivity across multiple serovars of C. trachomatis without host cell toxicity. The lead compound 21a exhibits reduced lipophilicity versus 1 and did not affect the growth or viability of representative commensal flora at 50 mu M. In microscopy studies, a highly active fluorescent analogue 37 localized inside the parasitiphorous inclusion, indicative of a specific targeting of bacterial components. In summary, we present a class of small molecules to enable the development of specific treatments for C. trachomatis.

  • 234.
    Gouveia-Figueira, Sandra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    Bosson, Jenny A.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Pulmonary Medicine.
    Unosson, Jon
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Pulmonary Medicine.
    Behndig, Annelie F.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Pulmonary Medicine.
    Nording, Malin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fowler, Christopher
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    Relative and absolute reliability of measures of linoleic acid-derived oxylipins in human plasma2015In: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 121, no Part B, p. 227-233Article in journal (Refereed)
    Abstract [en]

    Modern analytical techniques allow for the measurement of oxylipins derived from linoleic acid in biological samples. Most validatory work has concerned extraction techniques, repeated analysis of aliquots from the same biological sample, and the influence of external factors such as diet and heparin treatment upon their levels, whereas less is known about the relative and absolute reliability of measurements undertaken on different days. A cohort of nineteen healthy males were used, where samples were taken at the same time of day on two occasions, at least 7 days apart. Relative reliability was assessed using Lin's concordance correlation coefficients (CCC) and intraclass correlation coefficients (ICC). Absolute reliability was assessed by Bland-Altman analyses. Nine linoleic acid oxylipins were investigated. ICC and CCC values ranged from acceptable (0.56 [13-HODE]) to poor (near zero [9(10)- and 12(13)-EpOME]). Bland-Altman limits of agreement were in general quite wide, ranging from ±0.5 (12,13-DiHOME) to ±2 (9(10)-EpOME; log10 scale). It is concluded that relative reliability of linoleic acid-derived oxylipins varies between lipids with compounds such as the HODEs showing better relative reliability than compounds such as the EpOMEs. These differences should be kept in mind when designing and interpreting experiments correlating plasma levels of these lipids with factors such as age, body mass index, rating scales etc.

  • 235.
    Gouveia-Figueira, Sandra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Nording, Malin L.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Validation of a tandem mass spectrometry method using combined extraction of 37 oxylipins and 14 endocannabinoid-related compounds including prostamides from biological matrices2015In: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 121, no Part A, p. 110-121Article in journal (Refereed)
    Abstract [en]

    There is a clinical need for more relevant coverage of bioactive lipids using smaller sample volumes. Therefore, we have validated a tandem mass spectrometry method for combined solid phase extraction of 37 compounds in the oxylipin (OxL) and 14 in the endocannabinoid (eCB) metabolome, as well as prostamides. The limits of quantification (LOQ) for compounds in the eCB metabolome were in the range 0.5-1000fg on column, intraday accuracy and precision ranges (%) were 83-125 and 0.3-17, respectively, and interday accuracy and precision ranges (%) were 80-119 and 1.2-20, respectively, dependent upon the compound and the concentration studied. Corresponding values for OxL were 0.5fg-4.2pg on column (LOQ), 85-115% (inter- and intraday accuracy) and <5% (precision). The combined extraction method was successfully applied to tissues, cell extracts, human plasma and milk samples. A deeper study of levels in elk, pig and cow brain, as well as cow heart and liver revealed tissue and species-specific elevation of eicosanoids: arachidonate diols, 20-HETE and 12(S)-HEPE (cow liver), LTB4 (cow brain), and monohydroxy metabolites (HETEs), epoxides and 5-oxo-ETE in elk brain, which might be caused by factors of stress and/or post-mortem reactions in the tissues.

  • 236.
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Protein tyrosine kinases and the regulation of signalling and adhesion in drosophila melanogaster2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In order to build a multi-cellular organism and to regulate cellular functions, cells need to communicate with each other, as well as tightly regulate their behaviour in response to environmental changes. For these purposes all eukaryotic cells express a large number of membrane spanning receptors that either themselves contain catalytic activity or via cytoplasmic effector enzymes, function to transmit “signals” from the cell exterior to induce appropriate responses within the cell. Protein tyrosine kinases (PTKs) are important signalling molecules, represented by the transmembrane receptor tyrosine kinases (RTKs) in addition to the cytoplasmic non-receptor PTKs, which alter cell behaviour by phosphorylating target proteins. An additional requirement for proper signalling and multicellular organisation is the adhesion between cells as well as adhesion of cells to the extracellular matrix (ECM).

    Adhesion between cells and the ECM is mainly mediated by the integrin family of cell surface receptors, which functions as a structural link between the ECM and the actin cytoskeleton as well as important centres for signalling. Mammalian studies have implicated the cytoplasmic Focal Adhesion Kinase (FAK), as a major transmitter of signalling emanating from integrins, regulating cell migration, survival, proliferation and differentiation. In our studies of the sole FAK family member in Drosophila, Fak56, we have concluded that the deletion of Fak56 from the fruit fly genome causes no obvious defects in integrin-mediated adhesion, migration or signalling in vivo. Consequently, in contrast to the embryonic lethality observed in mouse knockouts, Fak56 mutant flies are both viable and fertile. However, we do find a clear genetic interaction between Fak56 and Drosophila integrins. Additionally, overexpression studies indeed indicate Fak56 as a negative regulator of integrin adhesion, given that excess Fak56 protein phenocopies loss of integrin function, causing phenotypes such as muscle detachment and wing blistering.

    In Drosophila, as well as in mammals, FAK family proteins are highly abundant in the CNS and in our studies we have identified a requirement of Fak56 in synaptic transmission at neuromuscular junctions. Lack of Fak56 causes a weakening of action potential conduction, resulting in sensitivity to high-frequency mechanical and electrical stimulation, manifested by epileptic-like seizures and paralysis in Fak56 mutants, a phenotype known as Bang Sensitivity (BS) in flies. We also show that Fak56 phosphorylation is directly modulated in response to alterations in intracellular calcium levels, supporting a role for Fak56 in neurotransmission.

    Fak56 is directly activated by the Drosophila Anaplastic Lymphoma Kinase, DAlk, receptor which was identified in our lab. We characterised DAlk as a novel RTK that is expressed in the embryonic CNS and mesoderm where it drives activation of the ERK/MAPK pathway. Indeed, we found DAlk to ectopically induce protein tyrosine phosphorylation and specifically phosphorylation of ERK, resulting in autonomous cell transformation and uncontrolled tissue growth. Subsequently, we identified a requirement for DAlk function during Drosophila embryogenesis, where it displays an essential role in gut development. Specifically, we identified the secreted molecule Jelly belly (Jeb) as a ligand for DAlk and showed that Jeb-DAlk interaction activates an ERK-mediated signalling pathway essential for visceral muscle specification and fusion, and consequently formation of the gut.

    The potent ability of PTKs to regulate cell behaviour, together with the strong linkage between RTK dysregulation and tumour formation, renders the negative regulation of kinase activity an important area of research. We have identified the Drosophila homologue of Cbl-interacting protein of 85kDa, dCIN85, an adaptor molecule which in mammalian cells has shown involvement in RTK endocytosis and downregulation, as well as in the regulation of actin cytoskeleton dynamics. In the fruit fly, dCIN85 displays essential functions, given that dCIN85 loss of function mutants display a grand-child less phenotype. Generation of a dCIN85 antibody, together with isoform-specific transgenic flies, have allowed us to observe a punctuate localization pattern of the SH3-domain containing dCIN85 variants, representing Rab5-positive endosomal structures. This, in addition to the confirmation of a direct dCIN85-dCbl interaction, indicates an evolutionary conservation of dCIN85 function. Interestingly, dCIN85 co-localises with dRICH1, a Cdc42 specific RhoGAP, in differentiated photoreceptor cells in eye imaginal discs. This may imply a role for dCIN85 in the regulation of the specialised endocytic recycling processes required for the assembly/maintenance of tight junctions and establishment of cell polarity in epithelial tissues.

  • 237.
    Granlund, Irene
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Proteomic analysis of Arabidopsis thaliana2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A complete proteome analysis of the chloroplast stroma, using 2D-PAGE, from spinach and Arabidopsis was performed. To improve the identification of proteins a computer program named SPECLUST was used. In SPECLUST, peak masses that are similar in many spots cluster together because they originate from the same protein with different locations on the gel. Within this program peaks in a cluster can be investigated in detail by peaks-in-common, and the unidentified masses that differ between spots in a cluster could be caused by protein modifications, which was analysed further by MS/MS.

    The thylakoid is an internal membrane system in the chloroplast where protein complexes involved in photosynthesis are housed. Enclosed in the thylakoid membrane is the chloroplast lumen, with a proteome estimated to contain 80-200 different proteins. Because the chloroplast lumen is close to the photosynthesis machinery in the plant, one can expect that the lumen proteome will change depending on if the plant is dark or light adapted. DIGE analysis of lumen proteins found that 15 lumen proteins show increased relative abundance in light-adapted plants. In addition co-expression analysis of lumen protein genes suggests that the lumen protein genes are uniformly transcriptionally regulated, not only by light but in a general manner.

    Plastocyanin is one of the proteins involved in the electron transfer in photosynthesis. Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis, where PETE2 is the more abundant isoform. Knockout mutants of each of the plastocyanin isoforms shows that a 90% reduction of plastocyanin levels affects rates of photosynthesis and growth only slightly. A corresponding over-expression of plastocyanin in each of the two knockout mutants results in essentially wild-type photosynthetic performance. Reduced plastocyanin levels make the plant sensitive to Cu stress and therefore plastocyanin plays a major role as a Cu sink.

    A by-product of photosynthesis is hydrogen peroxide, which may be harmful for the plant. The discovery that an abundant protein found in the chloroplast lumen, TL29, shared sequence homology to Ascorbate Peroxidase (APX) was therefore of interest. We have evidence that TL29 is not an APX protein; it lacks the heme-binding active site and shows no activity. TL29 is located in the grana region and is electrostaticaly attached to the thylakoid membrane. It has four isoforms, with different pIs, both in the native and denatured form. It has no interaction with ascorbate, when compared to raAPX1. TL29 has two cysteine residues and one of them seems to have redox-regulated function, proposing that it may interact with other proteins close to PSII.

  • 238.
    Gratz, Regina
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Expression Studies of AminoAcid Transporters belonging to the Lysine and Hisitidine Transporter (LHT) Family in Hybrid Aspen Populus tremula L. x tremuloides Michx.2013Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The human based input of fixed nitrogen, e.g. due to nitrogenous fertilizers, is the second most important driver of global change. The active input was, however, necessary due to a fast growing demand for agricultural products in order to feed an expanding world population in the last decades. Severe environmental damages are visible now, which is why it is crucial to find alternative ways to increase plant growth and biomass production without applying massive amounts of fertilizers. One way is to identify genes, which are able to improve nitrogen use efficiency (NUE) in plants when manipulated. Especially genes involved in nitrogen uptake, assimilation and remobilization, such as amino acid transporters are of great interest. Therefore a detailed knowledge about molecular processes regarding nitrogen transport in the respective plant species is crucial. So far, there is not much known about amino acid uptake mechanisms in tree species, which is why this work focuses on hybrid aspen. It was aimed to investigate the tissue expression patterns of genes encoding putative amino acid transporters in order to find potential target genes for improving NUE in the long term.

    It was shown that eight homologs of a main Arabidopsis amino acid transporter, AtLHT1, are expressed in poplar. The eight amino acid transporters displayed different expression patterns, with expression in roots, stem and leaves of young hybrid aspen. To analyze the impacts of an increased amino acid uptake phenotype in a tree model system, PtLHT1.2 was cloned into an expression vector for Agrobacterium-mediated transformation into hybrid aspen. These results will be of great value for further studies regarding NUE in tree models.

  • 239. Griese, Julia J
    et al.
    Kositzki, Ramona
    Schrapers, Peer
    Branca, Rui M M
    Nordström, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lehtiö, Janne
    Haumann, Michael
    Högbom, Martin
    Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 42, p. 25254-25272Article in journal (Refereed)
    Abstract [en]

    Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn-II and Fe-II in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R.M., Lehtio , J., Graslund, A., Lubitz, W., Siegbahn, P. E., and Hogbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 1718917194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.

  • 240. Grosjean, Henri
    et al.
    de Crécy-Lagard, Valérie
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Aminoacylation of the anticodon stem by a tRNA-synthetase paralog: relic of an ancient code?2004In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 29, no 10, p. 519-522Article in journal (Refereed)
    Abstract [en]

    The activation and charging of amino acids onto the acceptor stems of their cognate tRNAs are the housekeeping functions of aminoacyl-tRNA synthetases. The availability of whole genome sequences has revealed the existence of synthetase-like proteins that have other functions linked to different aspects of cell metabolism and physiology. In eubacteria, a paralog of glutamyl tRNA synthetase, which lacks the tRNA-binding domain, was found to aminoacylate tRNA(Asp) not on the 3'-hydroxyl group of the acceptor stem but on a cyclopentene diol of the modified nucleoside queuosine present at the wobble position of anticodon loop. This modified nucleoside might be a relic of an ancient code.

  • 241.
    Gudey, Shyam Kumar
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Sundar, Reshma
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Mu, Yabing
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Wallenius, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Zang, Guangxiang
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Bergh, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Heldin, Carl-Henrik
    Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University.
    Landström, Marene
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University.
    TRAF6 stimulates the tumor-promoting effects of TGF beta type I receptor through polyubiquitination and activation of Presenilin 12014In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 7, no 307, article id ra2Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGF beta) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGF beta type I receptor (T beta RI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-alpha-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of T beta RI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a gamma-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGF beta increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the T beta RI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved T beta RI in the transmembrane domain between valine-129 and isoleucine-130, and ICD generation was inhibited when these residues were mutated to alanine. We also showed that, after entering the nucleus, T beta RI-ICD bound to the promoter and increased the transcription of the gene encoding T beta RI. The TRAF6- and PS1-induced intramembrane proteolysis of T beta RI promoted TGF beta-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the gamma-secretase inhibitor DBZ {(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b, d]azepin-7-yl)-propionamide}, generation of T beta RI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that gamma-secretase inhibitors may be useful for treating aggressive prostate cancer.

  • 242.
    Gunnarsson, David
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Reproductive toxicology of endocrine disruptors: effects of cadmium, phthalates and phytoestrogens on testicular steroidogenesis2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A number of investigations during the last two decades describe adverse trends in male reproductive health, which have been proposed to be caused by environmental factors with endocrine disrupting properties. In contrast to many other toxicants, endocrine disruptors often do not show linear dose-response relationships typical of those found in traditional toxicological studies. For many compounds, low-dose exposure causes effects opposite to the ones seen after high-dose exposure. In addition, the timing of exposure has been found to be critical. Hence, to correctly assess the impact of endocrine disruptors on reproductive health requires in-depth knowledge of their mechanisms of action.

    This thesis aimed at identifying the mechanisms underlying the effects of cadmium (Cd), phthalates and phytoestrogens on testicular steroidogenesis. For this purpose, in vitro as well as in vivo models were used. Cd was found to inhibit testosterone synthesis in vivo by down-regulating LH receptor gene expression and reducing the testicular levels of cAMP and StAR protein. In addition, Cd caused a pronounced increase in testicular prostaglandin F (PGF), suggesting that Cd exerts its suppressive effect on steroidogenesis also by inducing the inhibitory PKC pathway. Pre-treatment with zinc (Zn) protected completely against Cd-induced effects on testosterone and PGF. Furthermore, we observed that Cd exposure increased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in the testis. GAPDH is a potent coactivator of androgen receptor-mediated transcription and the up-regulation found in our study is probably a compensatory response to reduced testosterone concentrations. This finding is interesting since GAPDH has been proposed to have an important role in the regulation of apoptosis as well as sperm motility. We discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used phthalate di-(2-ethylhexyl) phthalate (DEHP), stimulates Leydig cell steroidogenesis in vitro, by a cAMP- and StAR-independent mechanism. MEHP exposure caused a similar effect in granulosa cells. Gene expression analysis revealed that MEHP is likely to stimulate steroidogenesis by increasing the amount of cholesterol available for steroid synthesis. In the last investigation, we examined the effects of low-dose phytoestrogen exposure on testosterone synthesis during puberty in male goats. Isoflavones present in clover increased plasma concentrations of testosterone and free as well as total triiodothyronine (T3). T3 has previously been shown to induce testosterone synthesis and it is possible that an elevated T3 secretion underlies the increased plasma testosterone levels.

    Reduced fertility and reproductive tract malformations affect both the individual and the society. Hence, a sound knowledge of reproductive toxicants is of crucial importance. The findings presented in this thesis provide new insights into the reproductive toxicology of endocrine disruptors and may be valuable for risk assessment purposes.

  • 243.
    Gupta, Arun A.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Reinartz, Ines
    Karunanithy, Gogulan
    Spilotros, Alessandro
    Jonna, Venkateswara Rao
    Hofer, Anders
    Svergun, Dmitri I.
    Baldwin, Andrew J.
    Schug, Alexander
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 18, Part B, p. 3157-3169Article in journal (Refereed)
    Abstract [en]

    Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.

  • 244. Gurvich, Olga L
    et al.
    Näsvall, S Joakim
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Baranov, Pavel V
    Björk, Glenn R
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Atkins, John F
    Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL2011In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 8, p. 3079-3092Article in journal (Refereed)
    Abstract [en]

    The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5’-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNA(Pro) mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting.

  • 245.
    Gussing, Fredrik
    Umeå University, Faculty of Medicine, Molecular Biology.
    Zonal organization of the mouse olfactory systems2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Animals survey their environment for relevant odorous chemical compounds by means of the olfactory system. This system is in most vertebrates divided into a main and accessory olfactory system with two specialized neuroepithelia, the olfactory and the vomeronasal epithelium, respectively. The sensory neurons reside in these epithelia and together the neurons have an extraordinary sensitivity and are capable of detecting a vast number of different chemical molecules. After processing the chemical information, behavior may be altered. The information about a chemicals structure is deconstructed into a format that the brain may process. This is facilitated by organizing sensory neurons into a map and that the individual neuron responds only to one chemical feature. The sensory maps appear to have zones with different neuronal subpopulations. This thesis is addressing the fact that establishment, maintenance and function of these zones are unknown.

    We identify a gene (NQO1) to be selectively expressed in defined zone of the olfactory and the vomeronasal epithelia, respectively. NQO1-positive and negative axons segregate within the olfactory nerve and maintain a zonal organization when reaching olfactory bulb target neurons. These results indicate that one zone of both the accessory and the main olfactory projection maps is composed of sensory neurons specialized in reducing environmental and/or endogenously produced quinones via an NQO1-dependent mechanism.

    In addition, we have identified genes expressed in a graded manner that correlates with the dorsomedial-ventrolateral zonal organization of the olfactory epithelia. Considering the known functions of identified genes in establishment of cell specificity and precise axonal targeting, we suggest that zonal division of the primary olfactory systems is maintained, during continuous neurogenesis, as a consequence of topographic counter gradients of positional information.

    The vomeronasal sensory neurons (VSN) are organized into an apical and a basal zone. The zones differ in expression of e.g. chemosensory receptor families and Gα protein subunits (Gαi2 and Gαo). We have analyzed transgenic mice (OMP-dnRAR) in which the VSNs are unresponsive to the function of one of the genes identified herein (RALDH2). The phenotype observed suggests that endogenous produced retinoic acid is selectively required for postnatal survival of neurons in the Gαo-positive zone. Analyses of another mouse line target deleted in the Gαi2 gene (Gαi2 mutant) reveal a cellular phenotype that is opposite to that of OMP-dnRAR mice. Consequently in these mice, the apical Gαi2-positive zone is reduced whereas VSNs in the basal zone are not affected.

    Several social and reproductive behaviors are under the influence of the vomeronasal organ. We have analyzed some behavioral consequences of having deficient neurons that corresponds to either of the two zones. We propose that cues important for aggressive behavior are detected by apical vomeronasal zone, while cues detected by both apical and basal VSNs influence gender preference behavior.

  • 246.
    Gustafson, Inga
    Umeå University, Faculty of Science and Technology, Chemistry.
    Phospholipid membranes in biosensor applications: Stability, activity and kinetics of reconstituted proteins and glycolipids in supported membranes2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In this study the formation of supported membranes onto planar solid supports has been investigated. The stability and activity of reconstituted membrane receptors has been studied. The potential use of such preparations in biosensor applications is discussed.

    The lipid films were made by the Langmuir Blodgett and by the liposome fusion techniques. These supported films were characterised by ellipsometry, atomic force microscopy, surface plasmon resonance (SPR) and resonant mirror techniques. The thickness of the films was in agreement with that of a cell membrane. The kinetics of formation of the lipid films was studied and discussed.

    The proteins, bacteriorhodopsin, cytochrome oxidase, acetylcholinesterase and the nicotinic acetylcholine receptor were reconstituted into the supported membrane. The subsequent analysis showed that the proteins were individually distributed and that the activity was retained, in some cases for several weeks after immobilisation.

    The glycolipids, GM1, GM2, GD1b, asialo-GM1, globotriaosylceramide, lactosylceramide and galactosylceramide, were also reconstituted into the supported membranes. Their specific interaction with the toxin ricin or with its B-chain was examined using SPR. The affinity of intact toxin and of its B-chain differed markedly and was pH dependent. The carbohydrate chain length and charge density of the glycolipids also influenced the affinity.

  • 247. Gustafsson, Robert
    et al.
    Berntsson, Ronnie P.-A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Martínez-Carranza, Markel
    El Tekle, Geniver
    Odegrip, Richard
    Johnson, Eric A.
    Stenmark, Pål
    Crystal structures of OrfX2 and P47 from a Botulinum neurotoxin OrfX-type gene cluster2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 22, p. 3781-3792Article in journal (Refereed)
    Abstract [en]

    Botulinum neurotoxins are highly toxic substances and are all encoded together with one of two alternative gene clusters, the HA or the OrfX gene cluster. Very little is known about the function and structure of the proteins encoded in the OrfX gene cluster, which in addition to the toxin contains five proteins (OrfX1, OrfX2, OrfX3, P47, and NTNH). We here present the structures of OrfX2 and P47, solved to 2.1 and 1.8 angstrom, respectively. We show that they belong to the TULIP protein superfamily, which are often involved in lipid binding. OrfX1 and OrfX2 were both found to bind phosphatidylinositol lipids.

  • 248.
    Gustavsson, Anna
    Umeå University, Faculty of Medicine, Molecular Biology.
    Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands.

    The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins.

    We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

  • 249. Gutierrez, Laurent
    et al.
    Mongelard, Gaelle
    Flokova, Kristyna
    Pacurar, Daniel I.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Novak, Ondrej
    Staswick, Paul
    Kowalczyk, Mariusz
    Pacurar, Monica
    Demailly, Herve
    Geiss, Gaia
    Bellini, Catherine
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Auxin Controls Arabidopsis Adventitious Root Initiation by Regulating Jasmonic Acid Homeostasis2012In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 24, no 6, p. 2515-2527Article in journal (Refereed)
    Abstract [en]

    Vegetative shoot-based propagation of plants, including mass propagation of elite genotypes, is dependent on the development of shoot-borne roots, which are also called adventitious roots. Multiple endogenous and environmental factors control the complex process of adventitious rooting. In the past few years, we have shown that the auxin response factors ARF6 and ARF8, targets of the microRNA miR167, are positive regulators of adventitious rooting, whereas ARF17, a target of miR160, is a negative regulator. We showed that these genes have overlapping expression profiles during adventitious rooting and that they regulate each other's expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160 and miR167. We demonstrate here that this complex network of transcription factors regulates the expression of three auxin-inducible Gretchen Hagen3 (GH3) genes, GH3.3, GH3.5, and GH3.6, encoding acyl-acid-amido synthetases. We show that these three GH3 genes are required for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and we demonstrate that they act by modulating jasmonic acid homeostasis. We propose a model in which adventitious rooting is an adaptive developmental response involving crosstalk between the auxin and jasmonate regulatory pathways.

  • 250. Gómez-Consarnau, Laura
    et al.
    Akram, Neelam
    Lindell, Kristoffer
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Pedersen, Anders
    Neutze, Richard
    Milton, Debra L
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    González, José M
    Pinhassi, Jarone
    Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation2010In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 8, article id e1000358Article in journal (Refereed)
    Abstract [en]

    Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean’s surface.

2345678 201 - 250 of 802
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf