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  • 301.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kuoppa, Kerstin
    FOI Swedish Defence Research Agency, Division of CBRN Defence and Security.
    Meibom, Karin L.
    Université Paris Descartes, Faculté de Médecine Necker-Enfants Malades; INSERM, U570, Unit of Pathogenesis of Systemic Infections.
    Alkhuder, Khaled
    Université Paris Descartes, Faculté de Médecine Necker-Enfants Malades; INSERM, U570, Unit of Pathogenesis of Systemic Infections.
    Dubail, Iharilalao
    Université Paris Descartes, Faculté de Médecine Necker-Enfants Malades; INSERM, U570, Unit of Pathogenesis of Systemic Infections.
    Dupuis, Marion
    Université Paris Descartes, Faculté de Médecine Necker-Enfants Malades; INSERM, U570, Unit of Pathogenesis of Systemic Infections.
    Charbit, Alain
    Université Paris Descartes, Faculté de Médecine Necker-Enfants Malades; INSERM, U570, Unit of Pathogenesis of Systemic Infections.
    Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis2009In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 5, p. 1866-80Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is a highly infectious pathogen that infects animals and humans, causing tularemia. The ability to replicate within macrophages is central for virulence and relies on expression of genes located in the Francisella pathogenicity island (FPI), as well as expression of other genes. Regulation of FPI-encoded virulence gene expression in F. tularensis involves at least four regulatory proteins and is not fully understood. Here we studied the RNA-binding protein Hfq in F. tularensis and particularly the role that it plays as a global regulator of gene expression in stress tolerance and pathogenesis. We demonstrate that Hfq promotes resistance to several cellular stresses (including osmotic and membrane stresses). Furthermore, we show that Hfq is important for the ability of the F. tularensis vaccine strain LVS to induce disease and persist in organs of infected mice. We also demonstrate that Hfq is important for stress tolerance and full virulence in a virulent clinical isolate of F. tularensis, FSC200. Finally, microarray analyses revealed that Hfq regulates expression of numerous genes, including genes located in the FPI. Strikingly, Hfq negatively regulates only one of two divergently expressed putative operons in the FPI, in contrast to the other known regulators, which regulate the entire FPI. Hfq thus appears to be a new pleiotropic regulator of virulence in F. tularensis, acting mostly as a repressor, in contrast to the other regulators identified so far. Moreover, the results obtained suggest a novel regulatory mechanism for a subset of FPI genes.

  • 302.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Salomonsson, Emelie
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Goloviov, Igor
    Umeå University, Faculty of Medicine, Department of 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, Department of Clinical Microbiology, Clinical Bacteriology.
    Forsberg, Åke
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    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.

  • 303.
    Forslund, Anna-Lena
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Salomonsson, Emelie Näslund
    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).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kuoppa, Kerstin
    Michell, Stephen
    Titball, Richard
    Oyston, Petra
    Noppa, Laila
    FOI.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis2010In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 10, p. 227-Article in journal (Refereed)
    Abstract [en]

    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.

  • 304.
    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). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Aili, Margareta
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wiklund, Magda-Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    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). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD2000In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 38, no 1, p. 85-102Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

  • 305.
    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.

  • 306. Fransson, Susanne
    et al.
    Hansson, Magnus
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. Univ Gothenburg, Sahlgrenska Acad, Dept Pathol, SE-40530 Gothenburg, Sweden.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Djos, Anna
    Berbegall, Ana
    Javanmardi, Niloufar
    Abrahamsson, Jonas
    Palmer, Ruth H.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Univ Gothenburg, Sahlgrenska Acad, Dept Med Chem & Cell Biol, SE-40530 Gothenburg, Sweden.
    Noguera, Rosa
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Univ Gothenburg, Sahlgrenska Acad, Dept Med Chem & Cell Biol, SE-40530 Gothenburg, Sweden.
    Kogner, Per
    Martinsson, Tommy
    Intragenic Anaplastic Lymphoma Kinase (ALK) Rearrangements: Translocations as a Novel Mechanism of ALK Activation in Neuroblastoma Tumors2015In: Genes, Chromosomes and Cancer, ISSN 1045-2257, E-ISSN 1098-2264, Vol. 54, no 2, p. 99-109Article in journal (Refereed)
    Abstract [en]

    Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis. 

  • 307. Fredholm, Simon
    et al.
    Willerslev-Olsen, Andreas
    Met, Özcan
    Kubat, Linda
    Gluud, Maria
    Mathiasen, Sarah L.
    Friese, Christina
    Blümel, Edda
    Petersen, David L.
    Hu, Tengpeng
    Nastasi, Claudia
    Lindahl, Lise M.
    Buus, Terkild B.
    Krejsgaard, Thorbjørn
    Wasik, Mariusz A.
    Kopp, Katharina L.
    Koralov, Sergei B.
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Bonefeld, Charlotte M.
    Geisler, Carsten
    Woetmann, Anders
    Iversen, Lars
    Becker, Jürgen C.
    Odum, Niels
    SATB1 in Malignant T Cells2018In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 138, no 8, p. 1805-1815Article in journal (Refereed)
    Abstract [en]

    Deficient expression of SATB1 hampers thymocyte development and results in inept T-cell lineages. Recent data implicate dysregulated SATB1 expression in the pathogenesis of mycosis fungoides, the most frequent variant of cutaneous T-cell lymphoma. Here, we report on a disease stage-associated decrease of SATB1 expression and an inverse expression of STAT5 and SATB1 in situ. STAT5 inhibited SATB1 expression through induction of microRNA-155. Decreased SATB1 expression triggered enhanced expression of IL-5 and IL-9 (but not IL-6 and IL-32), whereas increased SATB1 expression had the opposite effect, indicating that the microRNA-155 target SATB1 is a repressor of IL-5 and IL-9 in malignant T cells. In accordance, inhibition of STAT5 and its upstream activator JAK3 triggered increased SATB1 expression and a concomitant suppression of IL-5 and IL-9 expression in malignant T cells. In conclusion, we provide a mechanistic link between the proto-oncogenic JAK3/STAT5/microRNA-155 pathway, SATB1, and cytokines linked to CTCL severity and progression, indicating that SATB1 dysregulation is involved in cutaneous T-cell lymphoma pathogenesis.

  • 308. Fuchino, Katsuya
    et al.
    Bagchi, Sonchita
    Cantlay, Stuart
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wu, Di
    Bergman, Jessica
    Kamali-Moghaddam, Masood
    Flardh, Klas
    Ausmees, Nora
    Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 21, p. E1889-E1897Article in journal (Refereed)
    Abstract [en]

    Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

  • 309. Fuhrmann, Jakob
    et al.
    Schmidt, Andreas
    Spiess, Silvia
    Lehner, Anita
    Turgay, Kürsad
    Mechtler, Karl
    Charpentier, Emmanuelle
    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).
    Clausen, Tim
    McsB is a protein arginine kinase that phosphorylates and inhibits the heat-shock regulator CtsR2009In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 324, no 5932, p. 1323-1327Article in journal (Refereed)
    Abstract [en]

    All living organisms face a variety of environmental stresses that cause the misfolding and aggregation of proteins. To eliminate damaged proteins, cells developed highly efficient stress response and protein quality control systems. We performed a biochemical and structural analysis of the bacterial CtsR/McsB stress response. The crystal structure of the CtsR repressor, in complex with DNA, pinpointed key residues important for high-affinity binding to the promoter regions of heat-shock genes. Moreover, biochemical characterization of McsB revealed that McsB specifically phosphorylates arginine residues in the DNA binding domain of CtsR, thereby impairing its function as a repressor of stress response genes. Identification of the CtsR/McsB arginine phospho-switch expands the repertoire of possible protein modifications involved in prokaryotic and eukaryotic transcriptional regulation.

  • 310. Fulton, Joel
    et al.
    Mazumder, Bismoy
    Whitchurch, Jonathan B.
    Monteiro, Cintia J.
    Collins, Hilary M.
    Chan, Chun M.
    Clemente, Maria P.
    Hernandez-Quiles, Miguel
    Stewart, Elizabeth A.
    Amoaku, Winfried M.
    Moran, Paula M.
    Mongan, Nigel P.
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of Experimental Cancer Research, Department of Translational Medicine, Lund University, Clinical Research Centre, Malmö, Sweden.
    Ali, Simak
    Heery, David M.
    Heterodimers of photoreceptor-specific nuclear receptor (PNR/NR2E3) and peroxisome proliferator-activated receptor-gamma (PPAR gamma) are disrupted by retinal disease-associated mutations2017In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 8, article id e2677Article in journal (Refereed)
    Abstract [en]

    Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the nuclear receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand-binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of peroxisome proliferator-activated receptor-gamma (PPAR gamma)/NR1C3 and thyroid hormone receptor b (TRb) TR beta/NR1A2. The binding of PNR to PPAR. was specific for this paralog, as no interaction was observed with the LBDs of PPAR alpha/NR1C1 or PPAR delta/NR1C2. In support of these findings, PPAR. and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPAR. LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPAR gamma complex formation. Wild-type PNR, but not a PNR309G mutant, was able to repress PPAR gamma-mediated transcription in reporter assays. In summary, our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPAR. and TR beta that have potential importance in retinal development and disease.

  • 311.
    Fällman, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Jass, Jana
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dynamic properties of bacterial pili measured by optical tweezers2004In: Proceedings of SPIE - The International Society for Optical Engineering vol. 5514: Optical Trapping and Optical Micromanipulation, 2004, p. 763-773Conference paper (Refereed)
  • 312.
    Fällman, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Jass, Jana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion2004In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 19, no 11, p. 1429-1437Article in journal (Refereed)
    Abstract [en]

    An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle’s displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads

  • 313.
    Fällman, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Jass, Jana
    Department of Microbiology and Immunology, The Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics.
    The unfolding of the P pili quaternary structure by stretching is reversible, not plastic2005In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 6, no 1, p. 52-56Article in journal (Refereed)
    Abstract [en]

    P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of ∼103PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.

  • 314.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Deleuil, Fabienne
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    McGee, Karen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Resistance to phagocytosis by Yersinia2002In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 291, no 6-7, p. 501-509Article in journal (Refereed)
    Abstract [en]

    Enteropathogenic species of the genus Yersinia penetrate the intestinal epithelium and then spread to the lymphatic system, where they proliferate extracellularly. At this location, most other bacteria are effectively ingested and destroyed by the resident phagocytes. Yersinia, on the other hand binds to receptors on the external surface of phagocytes, and from this location it blocks the capacity of these cells to exert their phagocytic function via different receptors. The mechanism behind the resistance to phagocytosis involves the essential virulence factor YopH, a protein tyrosine phosphatase that is translocated into interacting target cells via a type III secretion machinery. YopH disrupts peripheral focal complexes of host cells, seen as a rounding up of infected cells. The focal complex proteins that are dephosphorylated by YopH are focal adhesion kinase and Crk-associated substrate, the latter of which is a common substrate in both professional and non-professional phagocytes. In macrophages additional substrates have been found, the Fyn-binding/SLP-76-associated protein and SKAP-HOM. Phagocytosis is a rapid process that is activated when the bacterium interacts with the phagocyte. Consequently, the effect exerted by a microbe to block this process has to be rapid and precise. This review deals with the mechanisms involved in impeding uptake as well as with the role of the YopH substrates and focal complex structures in normal cell function.

  • 315.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gustavsson, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cellular mechanisms of bacterial internalization counteracted by Yersinia2005In: International Review of Cytology: a survey of cell biology / [ed] Kwang W. Jeon, Elsevier, 2005, Vol. 246, p. 135-188Chapter in book (Refereed)
    Abstract [en]

    Upon host-cell contact, human pathogenic Yersinia species inject Yop virulence effectors into the host through a Type III secretion-and-translocation system. These virulence effectors cause a block in phagocytosis (YopE, YopT, YpkA, and YopH) and suppression of inflammatory mediators (YopJ). The Yops that block phagocytosis either interfere with the host cell actin regulation of Rho GTPases (YopE, YopT, and YpkA) or specifically and rapidly inactivate host proteins involved in signaling from the receptor to actin (YopH). The block in uptake has been shown to be activated following binding to Fc, Complement, and beta1-integrin receptors in virtually any kind of host cell. Thus, the use of Yersinia as a model system to study Yersinia-host cell interactions provides a good tool to explore signaling pathways involved in phagocytosis.

  • 316.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gustavsson, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia inhibition of phagocytosis2006In: Phagocytosis of bacteria and Bacterial Pathogenicity, Cambridge: Cambridge University Press, 2006, p. 181-218Chapter in book (Other academic)
  • 317.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Cathrine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schesser, K.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Bidirectional signaling between Yersinia and its target cell1998In: Folia microbiologica (Prague), ISSN 0015-5632, E-ISSN 1874-9356, Vol. 43, no 3, p. 263-273Article in journal (Refereed)
    Abstract [en]

    Preventing the early host immune defense allows pathogenic Yersinia to proliferate in lymphatic tissue. This ability depends on signaling that occurs between the bacteria and the host cells. Following intimate contact with the target cell a signal is generated within the bacterium that results in increased expression of virulence-associated proteins that are subsequently delivered into the infected cell. These proteins, designated Yops, interfere with the host-cell signaling pathways that are normally activated to eliminate infectious agents.

  • 318.
    Fällman, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Cathrine
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Yersinia proteins that target host cell signaling pathways1997In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 99, no 6, p. 1153-1157Article, review/survey (Refereed)
  • 319.
    Gaca, Anthony O.
    et al.
    Rochester, New York, USA .
    Kudrin, Pavel
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Colomer-Winter, Cristina
    Rochester, New York, USA .
    Beljantseva, Jelena
    University of Tartu, Institute of Technology, Tartu, Estonia.
    Liu, Kuanqing
    Madison, Wisconsin, USA .
    Anderson, Brent
    Madison, Wisconsin, USA .
    Wang, Jue D.
    Madison, Wisconsin, USA .
    Rejman, Dominik
    Prague, Czech Republic.
    Potrykus, Katarzyna
    Gdańsk, Poland; Bethesda, Maryland, USA.
    Cashel, Michael
    Bethesda, Maryland, USA.
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Tartu, Estonia.
    Lemos, Jose A.
    Rochester, New York, USA .
    From (p)ppGpp to (pp)pGpp: characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis2015In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 197, no 18, p. 2908-2919Article in journal (Refereed)
    Abstract [en]

    The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p) ppGpp. In Enterococcus faecalis, (p) ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (Rel(Ef)) and the small alarmone synthetase (SAS) RelQ(Ef). Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQ(Ef) synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p) ppGpp synthesis from GDP and GTP, RelQ(Ef) also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p) ppGpp. We found that pGpp, like (p) ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQ(Ef) was activated only by ppGpp. Furthermore, enzymatic activity of RelQ(Ef) is insensitive to relacin, a (p) ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p) ppGpp. IMPORTANCE Accumulation of the nucleotide second messengers (p) ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p) ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p) ppGpp synthetase RelQ of Enterococcus faecalis (RelQ(Ef)), we found that, in addition to (p) ppGpp, RelQ(Ef) is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p) ppGpp. These findings provide a new regulatory feature of RelQ(Ef) and suggest that pGpp may represent a new member of the (pp) pGpp family of alarmones.

  • 320. Galka, Frank
    et al.
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Kusch, Harald
    Engelmann, Susanne
    Hecker, Michael
    Schmeck, Bernd
    Hippenstiel, Stefan
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Steinert, Michael
    Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.2008In: Infection and immunity, ISSN 1098-5522, Vol. 76, no 5, p. 1825-36Article in journal (Refereed)
    Abstract [en]

    Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

  • 321. Gallio, Marco
    et al.
    Englund, Camilla
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kylsten, Per
    Samakovlis, Christos
    Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline.2004In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 131, no 15, p. 3605-3614Article in journal (Refereed)
    Abstract [en]

    EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. We present evidence that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing it. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. We propose that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline.

  • 322. Garcia-Aljaro, Cristina
    et al.
    Melado-Rovira, Silvia
    Milton, Debra L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Blanch, Anicet R.
    Quorum-sensing regulates biofilm formation in Vibrio scophthalmi2012In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, p. 287-Article in journal (Refereed)
    Abstract [en]

    Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits.

    Results: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study.

    Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

  • 323.
    Gekara, Nelson O.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    DNA damage-induced immune response: Micronuclei provide key platform2017In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, no 10, p. 2999-3001Article in journal (Other academic)
    Abstract [en]

    DNA damage-induced activation of the cytoplasmic DNA sensor cGAS influences the outcome of infections, autoinflammation, and cancer. Recent studies by Harding et al. (2017. Nature. http://dx.doi.org/10.1038/nature23470), Mackenzie et al. (2017. Nature. http://dx.doi.org/10.1038/nature23449), and Bartsch et al. (2017. Human Molecular Genetics. https://doi.org/10.1093/hmg/ddx283) demonstrate a role for micronuclei formation in DNA damage-induced immune activation.

  • 324.
    Gerpe, M.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Kling, P.
    Berg, A. H.
    Olsson, P.-E.
    Arctic char (Salvelinus alpinus) metallothionein: cDNA sequence, expression, and tissue-specific inhibition of cadmium-mediated metallothionein induction by 17ß-estradiol, 4-OH-PCB 30, and PCB 1042000In: Environmental Toxicology and Chemistry, ISSN 0730-7268, E-ISSN 1552-8618, Vol. 19, no 3, p. 638-645Article in journal (Refereed)
  • 325. 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.

  • 326.
    Goldsteins, Gundars
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Karin
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dacklin, Ingrid
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edvinsson, Åsa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Sandgren, Ola
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Thylén, Christina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Characterisation of two highly amyloidogenic mutants of transthyretin1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, no 18, p. 5346-5352Article in journal (Refereed)
    Abstract [en]

    The plasma protein transthyretin (TTR) has the potential to form amyloid under certain conditions. More than 50 different point mutations have been associated with amyloid formation that occurs only in adults. It is not known what structural changes are introduced into the structure of this otherwise stable molecule that results in its aggregation into insoluble amyloid fibrils. On the basis of calculations of the frequency of known mutations over the polypeptide, we have constructed two mutants in the D-strand of the polypeptide. These molecules, containing either a deletion or a substitution at amino acid positions 53−55, were unstable and spontaneously formed aggregates upon storage in TBS (pH 7.6). The precipitates were shown to be amyloid by staining with thioflavin T and Congo Red. Their ultrastructure was very similar to that of amyloid fibrils deposited in the vitreous body of patients with familial amyloidotic polyneuropathy type 1 with an amino acid replacement in position 30 (TTRmet30). Like amyloid isolated from the vitreous body of the eye, the amyloid precipitates generated from the TTR mutants exposed a trypsin cleavage site between amino acid residues 48 and 49, while plasma TTRmet30 isolated from amyloidosis patients as well as wild-type TTR only showed minor trypsin sensitivity. Our data indicate that the mutants we have constructed are similar to amyloid precursors or may share structural properties with intermediates on a pathway leading to amyloid deposits of plasma TTR.

  • 327.
    Goldsteins, Gundars
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Persson, Håkan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Karin
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dacklin, Ingrid
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edvinsson, Åsa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saraiva, Maria João
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Exposure of cryptic epitopes on transthyretin only in amyloid and in amyloidogenic mutants1999In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 6, p. 3108-3113Article in journal (Refereed)
    Abstract [en]

    The structural requirements for generation of amyloid from the plasma protein transthyretin (TTR) are not known, although it is assumed that TTR is partly misfolded in amyloid. In a search for structural determinants important for amyloid formation, we generated a TTR mutant with high potential to form amyloid. We demonstrated that the mutant represents an intermediate in a series of conformational changes leading to amyloid. Two monoclonal antibodies were generated against this mutant; each displayed affinity to ex vivo TTR and TTR mutants with amyloidogenic folding but not to wild-type TTR or mutants exhibiting the wild-type fold. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and which we propose is displaced at the initial phase of amyloid formation, opening up new surfaces necessary for autoaggregation of TTR monomers. The results provide direct biochemical evidence for structural changes in an amyloidogenic intermediate of TTR.

  • 328. Gomzikova, Marina O.
    et al.
    Zhuravleva, Margarita N.
    Miftakhova, Regina R.
    Arkhipova, Svetlana S.
    Evtugin, Vladimir G.
    Khaiboullina, Svetlana F.
    Kiyasov, Andrey P.
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mongan, Nigel P.
    Pestell, Richard G.
    Rizvanov, Albert A.
    Cytochalasin B-induced membrane vesicles convey angiogenic activity of parental cells2017In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 41, p. 70496-70507Article in journal (Refereed)
    Abstract [en]

    Naturally occurring extracellular vesicles (EVs) play essential roles in intracellular communication and delivery of bioactive molecules. Therefore it has been suggested that EVs could be used for delivery of therapeutics. However, to date the therapeutic application of EVs has been limited by number of factors, including limited yield and full understanding of their biological activities. To address these issues, we analyzed the morphology, molecular composition, fusion capacity and biological activity of Cytochalasin B-induced membrane vesicles (CIMVs). The size of these vesicles was comparable to that of naturally occurring EVs. In addition, we have shown that CIMVs from human SH-SY5Y cells contain elevated levels of VEGF as compared to the parental cells, and stimulate angiogenesis in vitro and in vivo.

  • 329.
    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.

  • 330.
    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.

  • 331. Goormaghtigh, Frederic
    et al.
    Fraikin, Nathan
    Putrins, Marta
    Hallaert, Thibaut
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Garcia-Pino, Abel
    Sjödin, Andreas
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Division of CBRN Security and Defence, FOI–Swedish Defence Research Agency, Umeå, Sweden.
    Kasvandik, Sergo
    Udekwu, Klas
    Tenson, Tanel
    Kaldalu, Niilo
    Van Melderen, Laurence
    Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 3, article id e00640-18Article in journal (Refereed)
    Abstract [en]

    Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

  • 332. Goormaghtigh, Frederic
    et al.
    Fraikin, Nathan
    Putrins, Marta
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Garcia-Pino, Abel
    Udekwu, Klas
    Tenson, Tanel
    Kaldalu, Niilo
    Van Melderen, Laurence
    Reply to Holden and Errington, "Type II Toxin-Antitoxin Systems and Persister Cells"2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 5, article id e01838-18Article in journal (Refereed)
  • 333.
    Grabbe, Caroline
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Husnjak, Koraljka
    Dikic, Ivan
    The spatial and temporal organization of ubiquitin networks2011In: Nature reviews. Molecular cell biology, ISSN 1471-0072, E-ISSN 1471-0080, Vol. 12, no 5, p. 295-307Article in journal (Refereed)
    Abstract [en]

    In the past decade, the diversity of signals generated by the ubiquitin system has emerged as a dominant regulator of biological processes and propagation of information in the eukaryotic cell. A wealth of information has been gained about the crucial role of spatial and temporal regulation of ubiquitin species of different lengths and linkages in the nuclear factor-κB (NF-κB) pathway, endocytic trafficking, protein degradation and DNA repair. This spatiotemporal regulation is achieved through sophisticated mechanisms of compartmentalization and sequential series of ubiquitylation events and signal decoding, which control diverse biological processes not only in the cell but also during the development of tissues and entire organisms.

  • 334.
    Gripenland, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Regulatory roles of two small RNAs in the human pathogen Listeria monocytogenes and the evaluation of an alternative infection model2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth.

    Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites.

    To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins.

    L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes.

    Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

  • 335.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Christopher
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Evaluating the chicken embryo as a model for studying Listeria monocytogenes pathogenesis: a role for the PrfA pathwayManuscript (preprint) (Other academic)
  • 336.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Andersson, Christopher
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Exploring the chicken embryo as a possible model for studying Listeria monocytogenes pathogenicity2014In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 4, article id 170Article in journal (Refereed)
    Abstract [en]

    Listeria monocytogenes is a bacterial pathogen capable of causing severe infections in humans, often with fatal outcomes. Many different animal models exist to study L. monocytogenes pathogenicity, and we have investigated the chicken embryo as an infection model: What are the benefits and possible drawbacks? We have compared a defined wild-type strain with its isogenic strains lacking well-characterized virulence factors. Our results show that wild-type L. monocytogenes, already at a relatively low infection dose (similar to 5 x 10(2) cfu), caused death of the chicken embryo within 36 h, in contrast to strains lacking the main transcriptional activator of virulence, PrfA, or the cytolysin LLO. Surprisingly, strains lacking the major adhesins InIA and InIB caused similar mortality as the wild-type strain. In conclusion, our results suggest that the chicken embryo is a practical model to study L. monocytogenes infections, especially when analyzing alternative virulence pathways independent of the InIA and InIB adhesins. However, the route of infection might be different from a human infection. The chicken embryo model and other Listeria infection models are discussed.

  • 337.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Dussurget, Olivier
    Institut Pasteur, Paris, France.
    Sesto, Nina
    Institut Pasteur, Paris, France..
    Byström, Jonas
    Queen Mary, University of London, London, Great Britain..
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bécavin, Christoph
    Institut Pasteur, Paris, France..
    Cossart, Pascale
    Institut Pasteur, Paris, France..
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rli38, a novel stress induced small RNA required for Listeria monocytogenes virulenceManuscript (preprint) (Other academic)
  • 338.
    Gripenland, Jonas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Netterling, Sakura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Loh, Edmund
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tiensuu, Teresa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Toledo-Arana, Alejandro
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    RNAs: regulators of bacterial virulence2010In: Nature Reviews Microbiology, ISSN 1740-1526, E-ISSN 1740-1534, Vol. 8, no 12, p. 857-866Article in journal (Refereed)
    Abstract [en]

    RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5' and 3' untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.

  • 339.
    Grundström, Christine
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Anjani
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Priya, Anshu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Negi, Neema
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    ETS1 and PAX5 transcription factors recruit AID to Igh DNA2018In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 48, no 10, p. 1687-1697Article in journal (Refereed)
    Abstract [en]

    B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

  • 340.
    Grundström, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Anjani
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Priya, Anshu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Regulation of diversification and affinity maturation of antibodies2016In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 38, p. S43-S43Article in journal (Other academic)
  • 341.
    Grundström, Thomas
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hauser, Jannek
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kumar, Ramesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ahmed, Tanzeel
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mechanisms controlling diversification and affinity maturation of antibodies2015In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 36, p. S43-S43Article in journal (Other academic)
  • 342. Grönholm, Juha
    et al.
    Kaustio, Meri
    Myllymäki, Henna
    Kallio, Jenni
    Saarikettu, Juha
    Kronhamn, Jesper
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Valanne, Susanna
    Silvennoinen, Olli
    Rämet, Mika
    Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells2012In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 3, p. 1239-1250Article in journal (Refereed)
    Abstract [en]

    The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-gamma- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.

  • 343. Guan, J.
    et al.
    Tucker, E. R.
    Wan, H.
    Chand, D.
    Danielson, L. S.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    El Wakil, A.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Witek, B.
    Jamin, Y.
    Umapathy, G.
    Robinson, S. P.
    Johnson, T. W.
    Smeal, T.
    Martinsson, T.
    Chesler, L.
    Palmer, R. H.
    Hallberg, B.
    The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN2016In: Disease Models and Mechanisms, ISSN 1754-8403, E-ISSN 1754-8411, Vol. 9, no 9, p. 941-952Article in journal (Refereed)
    Abstract [en]

    The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALK(F1174L)/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

  • 344.
    Guan, Jikui
    et al.
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    Yamazaki, Yasuo
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    Chand, Damini
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    van Dijk, Jesper R.
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Palmer, Ruth H.
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    Hallberg, Bengt
    Institute of Biomedicine,Dept of Medical Biochem and Cell Biol, Sahlgrenska Academy, University of Gothenburg..
    Novel mechanisms of ALK activation revealed by analysis of the Y1278S neuroblastoma mutation2017In: Cancers, ISSN 2072-6694, Vol. 9, no 11, article id 149Article in journal (Refereed)
    Abstract [en]

    Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

  • 345.
    Gunnarsson, David
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Leffler, Per
    Umeå University, Faculty of Science and Technology, European CBRNE Center.
    Ekwurtzel, Emelie
    Martinsson, Gunilla
    Liu, Kui
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Selstam, Gunnar
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes2008In: Reproduction, ISSN 1470-1626, E-ISSN 1476-3990, Vol. 135, no 5, p. 693-703Article in journal (Refereed)
    Abstract [en]

    Phthalates are widely used as plasticizers in a number of daily-life products. In this study, we investigated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the frequently used plasticizer di-(2-ethylhexyl) phthalate (DEHP), on gonadal steroidogenesis in vitro. MEHP (25–100 µM) stimulated basal steroid synthesis in a concentration-dependent manner in immortalized mouse Leydig tumor cells (MLTC-1). The stimulatory effect was also detected in KK-1 granulosa tumor cells. MEHP exposure did not influence cAMP or StAR protein levels and induced a gene expression profile of key steroidogenic proteins different from the one induced by human chorionic gonadotropin (hCG). Simultaneous treatment with MEHP and a p450scc inhibitor (aminoglutethimide) indicated that MEHP exerts its main stimulatory effect prior to pregnenolone formation. MEHP (10–100 µM) up-regulated hormone-sensitive lipase and 3-hydroxy-3-methylglutaryl coenzyme A reductase, suggesting that MEHP increases the amount of cholesterol available for steroidogenesis. Our data suggest that MEHP, besides its known inhibitory effect on hCG action, can directly stimulate gonadal steroidogenesis in both sexes through a cAMP- and StAR-independent mechanism. The anti-steroidogenic effect of DEHP has been proposed to cause developmental disorders such as hypospadias and cryptorchidism, whereas a stimulation of steroid synthesis may prematurely initiate the onset of puberty and theoretically affect the hypothalamic–pituitary–gonadal axis.

  • 346.
    Gunnarsson, David
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nordberg, Gunnar
    Umeå University, Faculty of Medicine, Public Health and Clinical Medicine.
    Lundgren, Per
    Umeå University, Faculty of Medicine, Clinical Sciences.
    Selstam, Gunnar
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats2003In: Toxicology, ISSN 0300-483X, E-ISSN 1879-3185, Vol. 183, no (1-3), p. 57-63Article in journal (Refereed)
    Abstract [en]

    Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose–response experiment Male Sprague–Dawley rats received a single subcutaneous (sc) injection of CdCl2 (1, 5 or 10 μmol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 μmol/kg). In the temporal–response experiment rats were given 10 μmol/kg of CdCl2 sc and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments.

  • 347.
    Gunnarsson, David
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Nordberg, Gunnar
    Umeå University, Faculty of Medicine, Public Health and Clinical Medicine, Occupational and Enviromental Medicine.
    Selstam, Gunnar
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Differential effects of cadmium on the gene expression of seven-transmembrane-spanning receptors and GAPDH in the rat testis.2007In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, Vol. 168, no 1, p. 51-7Article in journal (Refereed)
    Abstract [en]

    Cadmium (Cd) is a widely spread toxicant with endocrine disrupting properties. Under experimental conditions it suppresses sex steroid synthesis in the male as well as the female. Testicular steroidogenesis is primarily regulated by gonadotropins, but is also influenced by catecholamines. We have previously shown that Cd exposure affects rat testosterone synthesis by down-regulating luteinizing hormone (LH) receptor mRNA expression. In this study, rats were given 10 micromol/kg Cd subcutaneously and sacrificed 0.48-144 h later. We investigated the effects of Cd on testicular gene expression of two adrenergic receptors. In addition, mRNA levels of the androgen-regulated house keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured. In contrast to the suppressive influence on LH receptor expression Cd lacked effect on the expression of alpha(1A)- and beta(2)-adrenergic receptors. GAPDH gene expression, on the other hand, was up-regulated 1.6-fold after exposure to 10 micromol/kg Cd. These data suggest that the influence of Cd on testicular gene expression involves a specific effect on the LH receptor and not a general effect on seven-transmembrane-spanning receptors. Also, data indicate that the increased expression of GAPDH may be secondary to Cd-induced testosterone deprivation, suggesting future studies of androgen-regulated genes in the toxicity of Cd.

  • 348.
    Gunnarsson, David
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Selstam, Gunnar
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Ridderstråle, Yvonne
    Holm, Lena
    Ekstedt, Elisabeth
    Madej, Andrzej
    Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3) levels in male goat kids2009In: Acta Veterinaria Scandinavica, ISSN 1751-0147, E-ISSN 1751-0147, Vol. 51Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids. METHODS: Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3-4 mg/kg/day) for approximately 3 months. Plasma testosterone and total and free triiodothyronine (T3) concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA) was carried out. RESULTS: No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8) phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l). This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6). A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10) that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant. CONCLUSION: Our findings suggest that phytoestrogens can stimulate testosterone synthesis during puberty in male goats by increasing the secretion of T3; a hormone known to stimulate Leydig cell steroidogenesis. It is possible that feedback signalling underlies the tendency towards decreased steroid production at the end of the experiment.

  • 349.
    Gunnarsson, David
    et al.
    Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Svensson, Mona
    Umeå University, Faculty of Medicine, Public Health and Clinical Medicine, Occupational and Enviromental Medicine.
    Selstam, Gunnar
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Nordberg, Gunnar
    Umeå University, Faculty of Medicine, Public Health and Clinical Medicine, Occupational and Enviromental Medicine.
    Pronounced induction of testicular PGF2alpha and suppression of testosterone by cadmium: prevention by zinc2004In: Toxicology, ISSN 0300-483X, E-ISSN 1879-3185, Vol. 200, no 1, p. 49-58Article in journal (Refereed)
    Abstract [en]

    In order to investigate the effects of cadmium (Cd) on testicular prostaglandin F(PGF) production, adult male Sprague–Dawley rats were exposed to CdCl2 by subcutaneous injections. Dose–response as well as temporal–response experiments were performed, and PGF levels were determined by radioimmunoassay (RIA). The highest cadmium dose (10 μmol/kg) caused a dramatic elevation of testicular PGF, which was established to occur 48 h after exposure. At this point of time, cadmium-treated animals displayed PGF levels 16.7 times higher than saline-injected controls. No significant differences were found with the lower doses used (1 and 5 μmol/kg). In addition, the influence of pre-treatment with zinc (Zn) was assessed. The very strong stimulatory effect on PGF synthesis (22.3-fold) detected after exposure to 20 μmol/kg cadmium, was completely absent in the group given zinc (1 mmol/kg) prior to cadmium exposure. Plasma testosterone concentrations were determined in the three experiments, and all groups with strongly elevated PGF levels showed drastically lowered concentrations of testosterone. Zinc pre-treatment abolished not only the cadmium-induced rise in PGF but also the testosterone reduction. Additionally, cadmium was found to inhibit the expression of steroidogenic acute regulatory protein (StAR), which is responsible for the rate-limiting step in steroidogenesis. The present findings establish that cadmium can cause a strong induction of testicular PGF production, which might help to explain the well-known antisteroidogenic effect of this heavy metal. Such an inhibitory effect could be due to reduced levels of StAR.

  • 350.
    Guo, Betty P
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Teneberg, Susann
    Münch, Robert
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Terunuma, Daiyo
    Hatano, Ken
    Matsuoka, Koji
    Angström, Jonas
    Borén, Thomas
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Relapsing fever Borrelia binds to neolacto glycans and mediates rosetting of human erythrocytes.2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 46, p. 19280-19285Article in journal (Refereed)
    Abstract [en]

    A hallmark of acute relapsing fever borreliosis is severe bacteremia. Some Borrelia species, such as B. duttonii and B. crocidurae, associate with erythrocytes and induce aggregation recognized as erythrocyte rosetting. Erythrocyte rosettes contribute to disease severity by increased tissue invasiveness (such as invasion of CNS and encephalitis), hemorrhaging, and reduced blood flow in affected microcapillaries. Here we report that relapsing fever Borrelia binds to neolacto (Galbeta4GlcNAcbeta3Galbeta4Glcbeta1)-carrying glycoconjugates that are present on human erythrocytes. This interaction is of low affinity but is compensated for by the multivalency of neo-lacto-oligosaccharides on the erythrocyte cell surface. Hence, the protein-carbohydrate interaction is dependent on multivalent neolacto-glycans to mediate binding.

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