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  • 101.
    Berg, Håkan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Teleost reproduction: Aspects of Arctic char (Salvelinus alpinus) oocyte growth and maturation2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems.

    All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels.

    It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char.

    In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors.

    Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

  • 102.
    Berg, Håkan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Department of Marine Science, University of Texas Marine Science Institute, University of Texas, Port Aransas, Texas, USA.
    Olsson, Per-Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Department of Natural Science, Unit of Molecular Biology, Orebro University, Orebro, Sweden.
    Modig, Carina
    17ß-estradiol induced vitellogenesis is inhibited by cortisol at the post-transcriptional level in Arctic char (Salvelinus alpinus)2004Ingår i: Reproductive Biology and Endocrinology, ISSN 1477-7827, E-ISSN 1477-7827, Vol. 2, nr 62, s. 1-10Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    This study was performed to investigate stress effects on the synthesis of egg yolk precursor, vitellogenin (Vtg) in Arctic char (Salvelinus alpinus). In particular the effect of cortisol (F) was determined since this stress hormone has been suggested to interfere with vitellogenesis and is upregulated during sexual maturation in teleosts. Arctic char Vtg was purified and polyclonal antibodies were produced in order to develop tools to study regulation of vitellogenesis. The Vtg antibodies were used to develop an enzyme-linked immunosorbent assay. The corresponding Vtg cDNA was cloned from a hepatic cDNA library in order to obtain DNA probes to measure Vtg mRNA expression. Analysis of plasma from juvenile Arctic char, of both sexes, exposed to different steroids showed that production of Vtg was induced in a dose dependent fashion by 17β-estradiol (E2), estrone and estriol. Apart from estrogens a high dose of F also upregulated Vtg. In addition, F, progesterone (P) and tamoxifen were tested to determine these compounds ability to modulate E2 induced Vtg synthesis at both the mRNA and protein level. Tamoxifen was found to inhibit E2 induced Vtg mRNA and protein upregulation. P did not alter the Vtg induction while F reduced the Vtg protein levels without affecting the Vtg mRNA levels. Furthermore the inhibition of Vtg protein was found to be dose dependent. Thus, the inhibitory effect of F on Vtg appears to be mediated at the post-transcriptional level.

  • 103. Berggren, Kristina
    et al.
    Vindebro, Reine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bergström, Claes
    Spoerry, Christian
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Persson, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fex, Tomas
    Kihlberg, Jan
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Luthman, Kristina
    3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS2012Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, nr 6, s. 2549-2560Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.

  • 104.
    Berghard, Anna
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hägglund, Anna-Carin
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Bohm, Staffan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Leif
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons2012Ingår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, nr 8, s. 3464-3472Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Inactivation of the LIM-homeodomain 2 gene (Lhx2) results in a severe defect in specification of olfactory sensory neurons (OSNs). However, the ramifications of lack of Lhx2-dependent OSN specification for formation of the primary olfactory pathway have not been addressed, since mutant mice die in utero. We have analyzed prenatal and postnatal consequences of conditionally inactivating Lhx2 selectively in OSNs. A cell-autonomous effect is that OSN axons cannot innervate their target, the olfactory bulb. Moreover, the lack of Lhx2 in OSNs causes unpredicted, non-cell-autonomous phenotypes. First, the olfactory bulb shows pronounced hypoplasia in adults, and the data suggest that innervation by correctly specified OSNs is necessary for adult bulb size and organization. Second, absence of an olfactory nerve in the conditional mutant reveals that the vomeronasal nerve is dependent on olfactory nerve formation. Third, the lack of a proper vomeronasal nerve prevents migration of gonadotropin-releasing hormone (GnRH) cells the whole distance to their final positions in the hypothalamus during embryo development. As adults, the conditional mutants do not pass puberty, and these findings support the view of an exclusive nasal origin of GnRH neurons in the mouse. Thus, Lhx2 in OSNs is required for functional development of three separate systems.—Berghard, A., Hägglund, A.-C., Bohm, S., and Carlsson, L. Lhx2-dependent specification of olfactory sensory neurons is required for successful integration of olfactory, vomeronasal, and GnRH neurons.

  • 105.
    Bergman, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A sub-phenotype approach to dissect the genetic control of murine type 1 diabetes2002Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The non-obese diabetic (NOD) mouse is a model for human type 1 diabetes (T1D). The disease in the NOD mouse is polygenic and multifactorial and so far at least 20 insulin dependent diabetes (Idd) susceptibility loci have been identified. However, no etiological mutations have been definitely ascribed to the Idd loci. To identify potential etiological mutations, a sub-phenotype approach was undertaken, consisting of the establishment and genetic mapping of immuno-related sub-phenotypes that may contribute to the pathogenesis of T1D in the NOD mouse model. This thesis presents (1) the results of the identification and genetic mapping of four novel NOD immuno-phenotypes to individual Idd loci, and (2) confirmation of these results by the generation and analysis of congenic strains covering those Idd regions.

    Evidence is provided that gene(s) within the Idd5 region control cyclophosphamide (CY)-induced apoptosis in peripheral lymphocytes and y-irradiation induced apoptosis in NOD thymocytes. Analysis of non-obese resistant (NOR) and NOD-Idd5 congenic mice reveal that CY-induced apoptosis in peripheral lymphocytes and y-irradiation induced apoptosis in thymocytes are controlled by a 20cM and a 6cM region, respectively, both containing the Idd5 region and including the immuno-regulatory Ctla4 gene. Additionally, CTLA4 is shown to be defectively up-regulated in activated NOD peripheral lymphocytes, and CTLA4-deficient mice show similar defects in T cell apoptosis induction. Taken together, these results suggest that a defective up-regulation of CTLA4 mediates apoptosis resistance, contributing to diabetes pathogenesis.

    Moreover, it is shown that gene(s) within the Idd6 region control low proliferation ofNOD immature thymocytes and resistance to dexamethazone-induced apoptosis in immature DP thymocytes. The decrease of diabetes incidence and the restoration of the apoptosis resistance phenotype in reciprocal Idd6 congenic strains further restrict the chromosomal region controlling the Idd6 locus as well as the locus controlling the apoptosis resistance phenotype. In fact, analysis of NOD-Idd6 congenic mice reveal that Dxm-induced apoptosis in thymocytes is controlled by the distal 3cM region of the Idd6 locus. As the thymic selection process is highly dependent on both proliferation and apoptosis, the hypothesis is raised that the Idd6 locus contributes to the pathogenesis of diabetes by altering thymic selection, resulting in an autoimmune prone peripheral T cell repertoire.

  • 106.
    Bergman, Marie-Louise
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    Cilio, Corrado M
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Endocrine Research Unit, Wallenberg Laboratory, Malmö University Hospital MAS, University of Lund, 205 02 Malmö Sweden.
    Penha-Gonçalves, Carlos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    Lamhamedi-Cherradi, Salah-Eddine
    INSERM U25, Hopital Necker, Paris, France.
    Löfgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Colucci, Francesco
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lejon, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Garchon, Henri-Jean
    INSERM U25, Hopital Necker, Paris, France.
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Instituto Gulbenkian de Ciencia, Oeiras, Portugal.
    CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice2001Ingår i: Journal of Autoimmunity, ISSN 0896-8411, E-ISSN 1095-9157, Vol. 16, nr 2, s. 105-113Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The genes conferring susceptibility to autoimmune (insulin-dependent) diabetes mellitus (IDDM) are, in most cases, not defined. Among the loci so far identified as associated with murine IDDM (Idd1-19), only the nature of Idd1 has been assessed. Here we show that thymocytes and peripheral lymphocytes of the non-obese diabetic (NOD) mouse are relatively resistant to apoptosis induced by gamma-irradiation. By linkage analysis of F2 progeny mice, we map this trait to a locus on chromosome 1 containing the Idd5 diabetes susceptibility region. By the use of congenic mice, we confirm the linkage data and map this locus to a 6 cM region on proximal chromosome 1. Ctla4, being localized in this chromosomal region and mediating crucial functions in T cell biology, is a logical candidate gene in the Idd5 susceptibility region. In line with this, we demonstrate that T cells from Ctla4(-/-)deficient mice show a similar resistance to gamma-irradiation-induced apoptosis as observed in the NOD mice. This reinforces the notion that CTLA-4 contributes to the pathogenesis of autoimmune diabetes.

  • 107.
    Bergman, Marie-Louise
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Penha-Gonçalves, Carlos
    Gulbenkian Institute for Science, Oeiras, Portugal, PT.
    Lejon, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Low rate of proliferation in immature thymocytes of the non-obese diabetic mouse maps to the Idd6 diabetes susceptibility region2001Ingår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 44, nr 8, s. 1054-1061Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aims/hypothesis: The non-obese diabetic (NOD) mouse spontaneously develops T-cell-dependent autoimmune diabetes. This mouse strain has a number of immune dysfunctions related to T-cell development but so far there are no available data on the proliferation of NOD immature thymocytes. We therefore studied the thymocyte proliferation in the NOD mouse in discrete stages of T-cell development.

    Methods: We depleted thymocytes in vivo and analysed thymocyte proliferation during the thymus recovery from depletion. We used co-segregation analysis and quantitative loci trait analysis to investigate the genetic control of proliferation impairments in NOD thymocytes.

    Results: Immature thymocytes of female NOD mice proliferate with a relatively low rate compared to non-autoimmune C57Bl/6 mice. This aberrant proliferation was most pronounced in CD4 /loCD8+ cells differentiating from the CD4CD8 to the CD4+CD8+ stage. A genetic mapping study using an F2 intercross between the NOD and the C57BL/6 strains showed that a major locus controlling this trait is linked to the insulin-dependent diabetes susceptibility locus Idd6.

    Conclusion/interpretation: Our results suggest that impairment of proliferation of immature thymocytes is one possible mechanism through which the Idd6 locus contributes to the pathogenesis of diabetes.

  • 108.
    Bergqvist, Ingela
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Eriksson, Maria
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Saarikettu, Juha
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Eriksson, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Corneliussen, Brit
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmberg, Dan
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development2000Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 30, nr 10, s. 2857-2863Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

  • 109.
    Bergström, Sven
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Normark, Johan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Microbiological features distinguishing Lyme disease and relapsing fever spirochetes2018Ingår i: Wiener Klinische Wochenschrift, ISSN 0043-5325, E-ISSN 1613-7671, Vol. 130, nr 15-16, s. 484-490Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The recent proposal of splitting the genus Borrelia into two genera in the newly formed family of Borreliaceae, i.aEuro<overline>e. Borrelia and Borreliella has motivated us to reflect upon how these organisms has been characterized and differentiated. This article therefore aims to take a closer look on the biology and virulence attributes of the two suggested genera, i.aEuro<overline>e. those causing Lyme borreliosis and relapsing fever borreliosis. Both genera have much in common with similar infection biological features. They are both characterized as bacterial zoonoses, transmitted by hematophagous arthropods with almost identical microbiological appearance. Nevertheless, a closer look at the genotypic and phenotypic characteristics clearly reveals several differences that might motivate the suggested split. On the other hand, a change of this well-established classification within the genus Borrelia might impose an economical burden as well as a great confusion in society, including medical and scientific societies as well as the general population.

  • 110.
    Bergström, Sven
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Zückert, Wolfram R
    Structure, function and biogenesis of the Borrelia cell envelope2010Ingår i: Borrelia, molecular biology, host interactions and pathogenesis / [ed] Eds DS Samuels and JD Radolf, Norfolk, UK: Caister Academic Press , 2010, s. 139-166Kapitel i bok, del av antologi (Övrigt vetenskapligt)
  • 111.
    Bernardo, Lisandro
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    On the role of ppGpp and DksA mediated control of σ54-dependent transcription2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

  • 112.
    Bernardo, Lisandro
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Johansson, Linda
    Solera, Dafne
    Skärfstad, Eleonore
    Szalewska-Palasz, Agnieszka
    Shingler, Victoria
    The role of the alarmone ppGpp and DksA in regulation of σ54-dependent transcription in Pseudomonas putidaManuskript (Övrigt vetenskapligt)
  • 113. Bernardo-Garcia, Noelia
    et al.
    Sánchez-Murcia, Pedro A.
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Martínez-Caballero, Siseth
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hermoso, Juan A.
    Gago, Federico
    Cold-induced aldimine bond cleavage by Tris in Bacillus subtilis alanine racemase2019Ingår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 17, nr 17, s. 4350-4358Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/ MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.

  • 114. Berry, Teeara
    et al.
    Luther, William
    Bhatnagar, Namrata
    Jamin, Yann
    Poon, Evon
    Sanda, Takaomi
    Pei, Desheng
    Sharma, Bandana
    Vetharoy, Winston R
    Hallsworth, Albert
    Ahmad, Zai
    Barker, Karen
    Moreau, Lisa
    Webber, Hannah
    Wang, Wenchao
    Liu, Qingsong
    Perez-Atayde, Antonio
    Rodig, Scott
    Cheung, Nai-Kong
    Raynaud, Florence
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Robinson, Simon P
    Gray, Nathanael S
    Pearson, Andrew DJ
    Eccles, Suzanne A
    Chesler, Louis
    George, Rani E
    The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma2012Ingår i: Cancer Cell, ISSN 1535-6108, E-ISSN 1878-3686, Vol. 22, nr 1, s. 117-130Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALKF1174L and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.

  • 115.
    Bianchi, Matteo
    et al.
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Niemiec, Maria J.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Siler, Ulrich
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Reichenbach, Janine
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Redundant ability of phagocytes to kill Aspergillus species: Reply2011Ingår i: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 128, nr 3, s. 687-688Artikel i tidskrift (Refereegranskat)
  • 116. Bianchi, Matteo
    et al.
    Niemiec, Maria J
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Siler, Ulrich
    Urban, Constantin F.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Reichenbach, Janine
    Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent2011Ingår i: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 127, nr 5, s. 1243-1252 e.7Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function.

    OBJECTIVE: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD.

    METHODS: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or alpha-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils.

    RESULTS: We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice.

    CONCLUSION: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.

  • 117. Bielig, H
    et al.
    Rompikuntal, Pramod Kumar
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Mitesh, Dongre
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Zurek, B
    Lindmark, B
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ramstedt, Madeleine
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kufer, T A
    University of Cologne.
    NOD-like receptor activation by outer-membrane vesicles (OMVs) from non-O1 non-O139 Vibrio cholerae is modulated by the quorum sensing regulator HapR2011Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 4, s. 1418-1427Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. siRNA-mediated gene knock-down revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain, leucine rich repeat containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum sensing regulator HapR which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs in infective conditions. This work thus identified a new facet of how Vibrio affects host immune responses and defines a role for the quorum sensing machinery in this process.

  • 118.
    Billker, Oliver
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    CRISPRing the elephant in the room2018Ingår i: Cell Host and Microbe, ISSN 1931-3128, E-ISSN 1934-6069, Vol. 24, nr 6, s. 754-755Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host & Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium.

  • 119.
    Birve, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Suppressor of zeste 12, a Polycomb group gene in Drosophila melanogaster; one piece in the epigenetic puzzle2003Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In multicellular organisms all cells in one individual have an identical genotype, and yet their bodies consist of many and very different tissues and thus many different cell types. Somehow there must be a difference in how genes are interpreted. So, there must be signals that tell the genes when and where to be active and inactive, respectively. In some instances a specific an expression pattern (active or inactive) is epigenetic; it is established and maintained throughout multiple rounds of cell divisions. In the developing Drosophila embryo, the proper expression pattern of e.g. the homeotic genes Abd-B and Ubx is to be kept active in the posterior part and silenced in the anterior. Properly silenced homeotic genes are crucial for the correct segmentation pattern of the fly and the Polycomb group (Pc-G) proteins are vital for maintaining this type of stable repression.

    As part of this thesis, Suppressor of zeste 12 (Su(z)12) is characterized as a Drosophila Pc-G gene. Mutations in the gene cause widespread misexpression of several homeotic genes in embryos and larvae. Results show that the silencing of the homeotic genes Abd-B and Ubx, probably is mediated via physical binding of SU(Z)12 to Polycomb Response Elements in the BX-C. Su(z)12 mutations are strong suppressors of position-effect-variegation and the SU(Z)12 protein binds weakly to the heterochromatic centromeric region. These results indicate that SU(Z)12 has a function in heterochromatin-mediated repression, which is an unusual feature for a Pc-G protein. The structure of the Su(z)12 gene was determined and the deduced protein contains a C2-H2 zinc finger domain, several nuclear localization signals, and a region, the VEFS box, with high homology to mammalian and plant homologues. Su(z)12 was originally isolated in a screen for modifiers of the zeste-white interaction and I present results that suggests that this effect is mediated through an interaction between Su(z)12 and zeste. I also show that Su(z)12 interact genetically with other Pc-G mutants and that the SU(Z)12 protein binds more than 100 euchromatic bands on polytene chromosomes. I also present results showing that SU(Z)12 is a subunit of two different E(Z)/ESC embryonic silencing complexes, one 1MDa and one 600 kDa complex, where the larger complex also contains PCL and RPD3.

    In conclusion, results presented in this thesis show that the recently identified Pc-G gene, Su(z)12, is of vital importance for correct maintenance of silencing of the developmentally important homeotic genes.

  • 120.
    Bitar, Aziz
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Immunologi/immunkemi.
    Aung, Kyaw Min
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Immunologi/immunkemi.
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hammarström, Marie-Louise
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Immunologi/immunkemi.
    Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 7212Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.

  • 121. Bjarnsholt, Thomas
    et al.
    Hoiby, Niels
    Donelli, Gianfranco
    Imbert, Christine
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Understanding biofilms: are we there yet?2012Ingår i: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 65, nr 2, s. 125-126Artikel i tidskrift (Övrigt vetenskapligt)
  • 122.
    Björk, Glenn R
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Jacobsson, Kerstin
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nilsson, Kristina
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Johansson, Marcus J O
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Byström, Anders S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Persson, Olof P
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    A primordial tRNA modification required for the evolution of life?2001Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, nr 1-2, s. 231-239Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.

  • 123.
    Björk, Glenn R
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Wikström, P M
    Byström, Anders S
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Prevention of translational frameshifting by the modified nucleoside 1-methylguanosine1989Ingår i: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 244, nr 4907, s. 986-989Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The methylated nucleoside 1-methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in all transfer RNAs (tRNAs) that read codons starting with C except in those tRNAs that read CAN codons. All of the three proline tRNA species, which read CCN codons in Salmonella typhimurium, have been sequenced and shown to contain m1G in position 37. A mutant of S. typhimurium that lacks m1G in its tRNA when grown at temperatures above 37 degrees C, has now been isolated. The mutation (trmD3) responsible for this methylation deficiency is in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. Therefore, the three proline tRNAs in the trmD3 mutant have an unmodified guanosine at position 37. Furthermore, the trmD3 mutation also causes at least one of the tRNAPro species to frequently shift frame when C's are present successively in the message. Thus, m1G appears to prevent frameshifting. The data from eubacteria apply to both eukaryotes and archaebacteria.

  • 124.
    Björnfot, Ann-Catrin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins2009Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 13, s. 4259-4267Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

  • 125.
    Blomberg, Jeanette
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Regulation of apoptosis during treatment and resistance development in tumour cells2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Induction of apoptosis is the most studied cell death process and it is a tightly regulated physiological event that enables elimination of damaged and unwanted cells. Apoptosis can be induced via activation of either the intrinsic or the extrinsic signalling pathway. The intrinsic pathway involves activation of the mitochondria by stress stimuli, whereas the extrinsic pathway is triggered by ligand induced activation of death receptors such as Fas. Apoptosis induction via Fas activation plays an important role in the function of cytotoxic T lymphocytes and in the control of immune cell homeostasis.

    Several studies have shown that anticancer therapies require functional cell death signalling pathways. Irradiation based therapy has been successful in treatment of several malignancies but the usage of high doses has been associated with side effects. Therefore, low dose therapies, that either is optimized for specific delivery or administrated in combination with other treatments, are promising modalities. However, in order to achieve high-quality effects of such treatments, the death effector mechanisms involved in tumour eradication needs to be further explored. Importantly, tumour cells frequently acquire resistance to apoptosis, which consequently allows tumour cells to escape from elimination by the immune system and/or treatment.

    Interferons constitute a large family of pleotrophic cytokines that are important for the immune response against viruses and other microorganisms. The interferon signalling pathway mediates transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. Interferon has successfully been used in therapy against some tumours. However, several drawbacks have been reported, such as reduced sensitivity to interferon during treatment.

    The aim of this thesis was to elucidate mechanisms that mediate resistance to death receptor or interferon induced apoptosis in human tumour cell models, as well as investigate what molecular events that underlie cell death following radiation therapy of tumour cells.

    In order to elucidate mechanisms involved in acquired resistance to Fas- or interferon-induced apoptosis, a Fas- and interferon-sensitive human cell line, U937, was subjected to conditions where resistance to either Fas- or interferon induced apoptosis was acquired. Characterization of the Fas resistant cells showed that multiple resistant mechanisms had been acquired. Reduced Fas expression and increased cFLIP expression, which is an inhibitor of death receptor signalling, were two important changes found. To further examine the importance of these two alterations, clones from the Fas resistant population were established. The reduced Fas expression was determined to account for the resistant phenotype in approximately 70% of the clones. In the Fas resistant clones with normal Fas expression, the importance of an increased amount of the cFLIP protein was confirmed with shRNA interference. A cross-resistance to death receptor induced apoptosis was detected in the interferon resistant variant, which illustrates that a connection between death receptor and interferon induced apoptosis exists. Notably, interferon resistant cells also contained increased cFLIP expression, which were determined to mediate resistance to both interferon and death receptor mediated apoptosis. Finally, when cell death induced by irradiation treatment was investigated in HeLa Hep2 cells we could demonstrate that cell death was mediated by centrosome hyperamplification and mitotic aberrations, which forced the cells into mitotic catastrophes and delayed apoptosis.

    In conclusion, we have described model systems where selection for resistance to Fas or interferon induced apoptosis generated a heterogeneous population, where several signalling molecules were altered. Furthermore, we have shown that a complex cell death network was activated by irradiation based therapy.

  • 126.
    Blomberg, Jeanette
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Höglund, Andreas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Eriksson, David
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Immunologi/immunkemi.
    Ruuth, Kristina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Jacobsson, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nilsson, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Lundgren, Erik
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line2011Ingår i: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, nr 8, s. 783-794Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

  • 127.
    Blomberg, Jeanette
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Ruuth, Kristina
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Jacobsson, Maria
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Höglund, Andreas
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Nilsson, Jonas A
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lundgren, Erik
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosis2009Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, nr 2, s. 497-508Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.

  • 128.
    Blomberg, Jeanette
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Ruuth, Kristina
    Santos, Daniel
    Lundgren, Erik
    Acquired resistance to Fas/CD95 ligation in U937 cells is associated with multiple molecular mechanisms2008Ingår i: Anticancer research, ISSN 0250-7005, Vol. 28, nr 2A, s. 593-600Artikel i tidskrift (Refereegranskat)
  • 129. Blumel, Edda
    et al.
    Willerslev-Olsen, Andreas
    Gluud, Maria
    Lindahl, Lise M.
    Fredholm, Simon
    Nastasi, Claudia
    Krejsgaard, Thorbjorn
    Surewaard, Bas G. J.
    Koralov, Sergei B.
    Hu, Tengpeng
    Persson, Jenny L.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Clinical Research Center, Lund University, Sweden.
    Bonefeld, Charlotte Menne
    Geisler, Carsten
    Iversen, Lars
    Becker, Juergen C.
    Andersen, Mads Hald
    Woetmann, Anders
    Buus, Terkild Brink
    Odum, Niels
    Staphylococcal alpha-toxin tilts the balance between malignant and non-malignant CD4+ T cells in cutaneous T-cell lymphoma2019Ingår i: Oncoimmunology, ISSN 2162-4011, E-ISSN 2162-402XArtikel i tidskrift (Refereegranskat)
    Abstract [en]

    Staphylococcus aureus is implicated in disease progression in cutaneous T-cell lymphoma (CTCL). Here, we demonstrate that malignant T cell lines derived from CTCL patients as well as primary malignant CD4+ T cells from Sézary syndrome patients are considerably more resistant to alpha-toxin-induced cell death than their non-malignant counterparts. Thus, in a subset of Sézary syndrome patients the ratio between malignant and non-malignant CD4+ T cells increases significantly following exposure to alpha-toxin. Whereas toxin-induced cell death is ADAM10 dependent in healthy CD4+ T cells, resistance to alpha-toxin in malignant T cells involves both downregulation of ADAM10 as well as other resistance mechanisms. In conclusion, we provide first evidence that Staphylococcus aureus derived alpha-toxin can tilt the balance between malignant and non-malignant CD4+ T cells in CTCL patients. Consequently, alpha-toxin may promote disease progression through positive selection of malignant CD4+ T cells, identifying alpha-toxin as a putative drug target in CTCL.

  • 130.
    Boal, Frédéric
    et al.
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Xuereb, Jean-Marie
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Kunduzova, Oksana
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Sansonetti, Philippe J.
    INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Payrastre, Bernard
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France; .
    Tronchére, Héléne
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment2016Ingår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, nr 4, s. 750-759Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1), a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

  • 131. Bobay, Benjamin G
    et al.
    Thompson, Richele J
    Milton, Debra L
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Southern Research Institute, Birmingham, AL, USA.
    Cavanagh, John
    Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum2014Ingår i: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 8, nr 1, s. 177-179Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.

  • 132. Bober, Marta
    et al.
    Mörgelin, Matthias
    Olin, Anders I
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Collin, Mattias
    The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen.2011Ingår i: PloS one, ISSN 1932-6203, Vol. 6, nr 5, s. e20345-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.

  • 133.
    Bonde, Mari
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Structure and Function of the Borrelia burgdorferi Porins, P13 and P662015Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Borrelia burgdorferi är en bakterie med många unika egenskaper som orsakar sjukdomen Lyme borrelios. Borrelia kan idag lätt behandlas med antibiotika om sjukdomen upptäcks i ett tidigt stadium. Det är först om sjukdomen tillåts fortgå som symptom som nervsmärta och ansiktsförlamning kan uppstå och dessutom vara svåra att koppla till en Borrelia-infektion. Multiresistenta bakterier har blivit en stor del av vår vardag och även om Borrelia-bakterierna idag inte är resistenta mot flertalet antibiotika är det kanske speciellt viktigt, innan det är för sent, med forskning som kan leda till upptäckter av unika angreppsställen för nya läkemedel.

    Målet med denna avhandling var att studera hur två Borrelia proteiner, P13 och P66, ser ut, är uppbyggda och även vilken funktion de har. Dessa proteiner är tänkbara vaccinkandidater eftersom de sitter i yttre membranet hos bakterierna och sticker ut på ytan mot våra värdceller, vilket gör att vi reagerar mot dem vid en infektion. P13 och P66 är också viktiga kanaler för bakterierna vid upptag av näringsämnen och byggstenar från omgivningen. Ämnen som bakterierna inte kan producera själva. Pga. denna funktion är P13 och P66 tänkbara proteiner för blockering med ett läkemedel som skulle förhindra bakterien från att föröka sig i och med att de förlorar möjligheten att tillgodogöra sig näring. Detta i sin tur skulle leda till att vårt eget immunförsvar hinner rensa undan bakterierna innan infektionen blivit för stor och vi blivit sjuka. P66 har förutom porin funktionen även en adhesions funktion när proteinet kan binda integriner som sitter på olika typer av celler i vår kropp, bl. a. immunceller och epitelceller i våra blodkärl och vävnader. Den integrin bindande funktionen är viktig för bakterierna vid en infektion eftersom det gör det möjligt för bakterierna att binda till våra celler. Ett steg som är viktigt för att de senare ska kunna ta sig ut från blodkärlen till våra vävnader.

    P13 och P66 visade sig kunna bilda stora proteinkomplex i ytter membranet hos bakterierna med en storlek på 300 kDa respektive 420 kDa. De är inga specifika poriner som bara kan transportera en viss typ av molekyl med t.ex. en viss laddning, utan kan ombesörja upptaget av många olika typer av ämnen. Eliminering av p66 orsakade att ett annat adhesionsprotein, uppreglerades. En omplacering av ett normalt cytoplasmatiskt lokaliserat chaperon-protein till ytter-membranet hos bakterierna kunde också ses i frånvaro av P66. Chaperonet GroEL har i andra bakterier, bl. a. Helicobacter pylori, bakterien som orsakar magsår, beskrivits som ett protein som kan förflytta sig till ytan av bakterierna och där ha en liknande funktion som P66, dvs. att binda extracellulära matrisprotein. Förändringen i uttryck av adhesionsproteinet och förflyttningen av chaperonet till membranet var en följd av p66-eliminering och mest troligt ett sätt för bakterierna att komplettera den förlorade integrinbindande funktionen av P66.

    Det har tidigare visats att poriner är involverade i skyddet mot osmotisk stress i andra bakterier. Denna funktion hos P13 och P66 i Borrelia kunde inte ses när bakterier utsattes för osmotisk stress med glycerol, som orsakar en form av membranstress. Däremot kunde vi med hjälp av transkriptomanalys se att Borrelia-bakterier uppreglerade transkriptionen av två paraloger till P13 vid hyper-osmotisk stress. Borrelia bakteriens användning av dessa paraloga proteiner har tidigare trotts ske enbart i frånvaro av ett funktionellt P13 protein. Nu visade det sig att P13-paraloger har en egen funktion även i närvaro av P13, nämligen att vara involverade i regleringen av hyperosmotisk stress och därmed skydda bakterierna i denna stressituation. Andra gener som påverkades av osmotisk stress med glycerol var gener för stressfaktorer och pumpar i inre membranet hos bakterien.

  • 134.
    Bonde, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Frost, Mikaela
    Jegerschöld, Caroline
    Karolinska Institutet, Sweden.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structural analysis of the B. burgdorferi integral outer membrane protein, P13, in lipid bilayer NanodiscsManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
  • 135.
    Bonde, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Östberg, Yngve
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bunikis, Ignas
    Uppsala University.
    Nyunt Wai, Sun
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Effects of osmotic stress in P13 and P66 deficient Borrelia burgdorferi mutantsManuskript (preprint) (Övrig (populärvetenskap, debatt, mm))
  • 136.
    Bonnedahl, Jonas
    et al.
    Högskolan i Kalmar.
    Broman, Tina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Waldenström, Jonas
    Högskolan i Kalmar.
    Palmgren, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Niskanen, Taina
    Olsen, Björn
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    In search of human-associated bacterial pathogens in Antarctic wildlife: report from six penguin colonies regularly visited by tourists.2005Ingår i: Ambio: A Journal of the Human Environment, ISSN 0044-7447, Vol. 34, nr 6, s. 430-2Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We investigated the potential role of Antarctic tourism in the introduction of human-associated pathogens into Antarctic wildlife. We collected and analyzed 233 fecal samples from eight bird species. The samples were collected at six localities on the Antarctic Peninsula, which often is visited by tourists. Every sample was investigated for pathogens of potential human origin: Campylobacter jejuni, Salmonella spp., and Yersina spp. None of these bacteria was found. Our data suggest that the tourism industry so far has achieved its goal of not introducing pathogens into the Antarctic region. There is, however, an urgent need to further investigate the situation in areas closer to permanent Antarctic settlements.

  • 137.
    Brodiazhenko, Tetiana
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Johansson, Marcus J. O.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Takada, Hiraku
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nissan, Tracy
    Hauryliuk, Vasili
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Institute of Technology, University of Tartu, Tartu, Estonia.
    Murina, Victoriia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems2018Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, artikel-id 3041Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

  • 138. Broglia, Laura
    et al.
    Materne, Solange
    Lecrivain, Anne-Laure
    Hahnke, Karin
    Le Rhun, Anaïs
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Max Planck Unit for the Science of Pathogens, Berlin, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany; Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
    RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B2018Ingår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 15, nr 10, s. 1336-1347Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

  • 139.
    Broman, Tina
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Waldenström, Jonas
    Dahlgren, D
    Carlsson, I
    Eliasson, I
    Olsen, Björn
    Umeå universitet, Medicinsk fakultet, Klinisk mikrobiologi, Infektionssjukdomar.
    Diversities and similarities in PFGE profiles of Campylobacter jejuni isolated from migrating birds and humans.2004Ingår i: Journal of Applied Microbiology, ISSN 1364-5072, Vol. 96, nr 4, s. 834-43Artikel i tidskrift (Refereegranskat)
  • 140. Browall, Sarah
    et al.
    Norman, Martin
    Tångrot, Jeanette
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Bioinformatics Infrastructure for Life Sciences, Computational Life Science Cluster.
    Galanis, Ilias
    Sjöstrom, Karin
    Dagerhamn, Jessica
    Hellberg, Christel
    Pathak, Anuj
    Spadafina, Tiziana
    Sandgren, Andreas
    Bättig, Patrick
    Franzén, Oscar
    Andersson, Björn
    Örtqvist, Åke
    Normark, Staffan
    Henriques-Normark, Birgitta
    Intraclonal Variations Among Streptococcus pneumoniae Isolates Influence the Likelihood of Invasive Disease in Children2014Ingår i: Journal of Infectious Diseases, ISSN 0022-1899, E-ISSN 1537-6613, Vol. 209, nr 3, s. 377-388Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background. Pneumococcal serotypes are represented by a varying number of clonal lineages with different genetic contents, potentially affecting invasiveness. However, genetic variation within the same genetic lineage may be larger than anticipated. Methods. A total of 715 invasive and carriage isolates from children in the same region and during the same period were compared using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Bacterial genome sequencing, functional assays, and in vivo virulence mice studies were performed. Results. Clonal types of the same serotype but also intraclonal variants within clonal complexes (CCs) showed differences in invasive-disease potential. CC138, a common CC, was divided into several PFGE patterns, partly explained by number, location, and type of temperate bacteriophages. Whole-genome sequencing of 4 CC138 isolates representing PFGE clones with different invasive-disease potentials revealed intraclonal sequence variations of the virulence-associated proteins pneumococcal surface protein A (PspA) and pneumococcal choline-binding protein C (PspC). A carrier isolate lacking PcpA exhibited decreased virulence in mice, and there was a differential binding of human factor H, depending on invasiveness. Conclusions. Pneumococcal clonal types but also intraclonal variants exhibited different invasive-disease potentials in children. Intraclonal variants, reflecting different prophage contents, showed differences in major surface antigens. This suggests ongoing immune selection, such as that due to PspC-mediated complement resistance through varied human factor H binding, that may affect invasiveness in children.

  • 141.
    Brännström, Kristoffer
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Molecular dissection of established and proposed members of the Op18/Stathmin family of tubulin binding proteins2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    My initial aim was a functional analysis of the conserved Op18/stathmin family of microtubule-regulators, which includes the ubiquitous cytosolic Op18 protein and the neural membrane-attached RB3 and SCG10 proteins. The solved X-ray structure has shown that these proteins form a complex with tubulin -heterodimers via two imperfect helical repeats, which result in two head-to-tail aligned heterodimers in a tandem-tubulin complex. We have analyzed GTP exchange and GTP hydrolysis at the two exchangeable GTP-binding sites (E-site) within the tandem-tubulin complex. A comparison of Op18, RB3 and SCG10 proteins indicates that Op18/Stathmin family proteins have evolved to maintain the two heterodimers in a configuration that restrains the otherwise potent GTPase productive interactions facilitated by the head-to-head alignment of heterodimers in protofilaments. We concluded from these studies that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.

    To understand the significance of the large differences in tubulin affinity of Op18, RB3 and SCG10, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell-surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. We showed that, in contrast to CD2-Op18, both the CD2-SCG10 and CD2-RB3 chimeras sequester tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels. However, all three CD2-chimeras, including the tubulin sequestration-incompetent CD2-Op18, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during the interphase, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules.

    Sm16/SmSLP (Stathmin-Like Protein) has been identified as a protein released during skin penetration of the Schistosoma mansoni parasite. This protein has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. However, our studies refuted any functional similarity with stathmin/Op18 and we found instead that Sm16/SmSLP is a lipid bilayer binding protein that is taken up by cells through endocytosis.

    To study immuno-modulatory properties of Sm16/SmSLP, we designed an engineered version with decreased aggregation propensity, thus facilitating expression and purification of a soluble Sm16 /SmSLP protein from the eukaryotic organism Pichia pastoris. Determination of the hydrodynamic parameters revealed that both the recombinant and native Sm16/SmSLP is a ~9-subunits oligomer. The recombinant protein was found to have no effect on T lymphocyte activation, cell proliferation or the basal level of cytokine production of whole human blood or monocytic cells. Interestingly, however, recombinant Sm16 was found to potently inhibit the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and Poly(I:C). Since Sm16 specifically inhibits degradation of the IRAK1 signaling protein in LPS stimulated monocytes, it seems likely that inhibition is exerted proximal to the TLR-complex.

  • 142.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Nilsson, Lina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lee, Cheng Choo
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Pamrén, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Scanning electron microscopy as a tool for evaluating morphology of amyloid structures formed on surface plasmon resonance chips2018Ingår i: Data in Brief, E-ISSN 2352-3409, Vol. 19, s. 1166-1170Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We demonstrate the use of Scanning Electron microscopy (SEM) in combination with Surface Plasmon Resonance (SPR) to probe and verify the formation of amyloid and its morphology on an SPR chip. SPR is a technique that measures changes in the immobilized weight on the chip surface and is frequently used to probe the formation and biophysical properties of amyloid structures. In this context it is of interest to also monitor the morphology of the formed structures. The SPR chip surface is made of a layer of gold, which represent a suitable material for direct analysis of the surface using SEM. The standard SPR chip used here (CM5-chip, GE Healthcare, Uppsala, Sweden) can easily be disassembled and directly analyzed by SEM. In order to verify the formation of amyloid fibrils in our experimental conditions we analyzed also in-solution produced structures by using Transmission Electron Microscopy (TEM). For further details and experimental findings, please refer to the article published in Journal of Molecular Biology, (Brännström K. et al., 2018) [1].

  • 143.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Islam, Tohidul
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The role of histidines in amyloid β fibril assembly2017Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, nr 8, s. 1167-1175Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Low pH has a strong stabilising effect on the fibrillar assembly of amyloid β, which is associated with Alzheimer's disease. The stabilising effect is already pronounced at pH 6.0, suggesting that protonation of histidines might mediate this effect. Through the systematic substitution of the three native histidines in Aβ for alanines, we have evaluated their role in fibril stability. Using surface plasmon resonance, we show that at neutral pH the fibrillar forms of all His-Ala variants are destabilised by a factor of 4-12 compared to wild-type Aβ. However, none of the His-Ala Aβ variants impair the stabilising effect of the fibril at low pH.

  • 144.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lindhagen-Persson, Malin
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gharibyan, Anna L.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Iakovleva, Irina
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vestling, Monika
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sellin, Mikael E.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Brännström, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Morozova-Roche, Ludmilla
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Forsgren, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A Generic Method for Design of Oligomer-Specific Antibodies2014Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 3, s. e90857-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e. g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the A beta peptide and alpha-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.

  • 145.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Segerman, Bo
    Gullberg, Martin
    Functional dissection of GTP hydrolysis and exchange within the ternary complex of tubulin heterodimers and Op18/stathmin family members2003Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 19, s. 16651-16657Artikel i tidskrift (Refereegranskat)
  • 146.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sellin, Mikael E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Holmfeldt, Per
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Brattsand, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Dermatologi och venereologi.
    Gullberg, Martin
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.2009Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, nr 3, s. 1144-1154Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

  • 147.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Klinisk neurovetenskap.
    Nilsson, Lina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pihl, Mathias
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The N-terminal Region of Amyloid β Controls the Aggregation Rate and Fibril Stability at Low pH Through a Gain of Function Mechanism2014Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 136, nr 31, s. 10956-10964Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Alzheimer's disease is linked to a pathological polymerization of the endogenous amyloid β-peptide (Aβ) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aβ fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (KD), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the KD, polymerization cannot occur. However, the KD for Aβ has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aβ amyloid forms in vivo has been a matter of debate. Using SPR, we found that the KD of Aβ dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aβ to polymerize within a picomolar concentration range that is close to the physiological Aβ concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aβ contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aβ concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

  • 148.
    Brännström, Kristoffer
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Olofsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Brattsand, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Dermatologi och venereologi.
    Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis2012Ingår i: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 393, nr 5, s. 369-377Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.

  • 149.
    Bröms, Jeanette E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Type III secretion- the various functions of the translocon operon in bacterial pathogenesis2004Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.

    Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.

    The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.

    Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.

  • 150.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Katrin E
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion2005Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, nr 22, s. 7738-7752Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

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