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  • 51.
    Jacobson, Ken
    et al.
    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, USA.
    Lee, Juliet
    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, USA.
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lipid flow in locomoting cells: Response1991In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 251, no 4991, p. 318-318Article in journal (Refereed)
    Abstract [en]

    n/a

  • 52.
    Jager, Edwin
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Immerstrand, Charlotte
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Biosensors and Bioelectronics. Linköping University, The Institute of Technology.
    Biomedical applications of polypyrrole microactuators: from single-cell clinic to microrobots2000In: 1st Annual International, Conference On Microtechnologies in Medicine and Biology. 2000, IEEE , 2000, p. 58-61Conference paper (Other academic)
    Abstract [en]

    Microtools that will be useful for the positioning and investigation microstructures must operate relevant environments, such as cell culture media or blood plasma. They must also be comparatively strong, and preferably allow a muscle like mode of movement. This is given by a novel family of actuators based on conjugated polymers (like polypyrrole, PPy). By miniaturising these structures using standard photolithographic techniques, the authors can reduce the size down to 10-micrometer dimensions and build mechanically active microdevices. These can be moved and positioned by applying a potential to dope or undope the PPy. These novel structures are now being developed as a unique microactuator technology, suitable for operation in applications coupled to cell biology and biomedicine

  • 53.
    Jager, Edwin W.H.
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    Immerstrand, Charlotte
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lundström, Ingemar
    Linköping University, Department of Physics, Chemistry and Biology, Applied Physics. Linköping University, The Institute of Technology.
    Inganäs, Olle
    Linköping University, Department of Physics, Chemistry and Biology, Biomolecular and Organic Electronics. Linköping University, The Institute of Technology.
    The cell clinic: closable microvials for single cell studies2002In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 4, no 3, p. 177-187Article in journal (Refereed)
    Abstract [en]

    We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.

  • 54.
    Jarmalaviciute, Akvile
    et al.
    State Research Institute Centre Innovat Med, Lithuania .
    Tunaitis, Virginijus
    State Research Institute Centre Innovat Med, Lithuania .
    Strainiene, Egle
    Vilnius Gediminas Technical University, Lithuania .
    Aldonyte, Ruta
    State Research Institute Centre Innovat Med, Lithuania .
    Ramanavicius, Arunas
    Vilnius State University, Lithuania .
    Venalis, Algirdas
    State Research Institute Centre Innovat Med, Lithuania .
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Pivoriunas, Augustas
    State Research Institute Centre Innovat Med, Lithuania .
    A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth2013In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, no 2, p. 307-317Article in journal (Refereed)
    Abstract [en]

    Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF beta-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.

  • 55.
    Karlsson, Thommie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Bolshakova, Anastasia
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magalhães, Marco A.O
    Faculty of Dentistry, University of Toronto, Toronto, Canada.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Fluxes of Water through Aquaporin 9 Weaken Membrane-Cytoskeleton Anchorage and Promote Formation of Membrane Protrusions2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4, p. e59901-Article in journal (Refereed)
    Abstract [en]

    All modes of cell migration require rapid rearrangements of cell shape, allowing the cell to navigate within narrow spaces in an extracellular matrix. Thus, a highly flexible membrane and a dynamic cytoskeleton are crucial for rapid cell migration. Cytoskeleton dynamics and tension also play instrumental roles in the formation of different specialized cell membrane protrusions, viz. lamellipodia, filopodia and membrane blebs. The flux of water through membrane-anchored water channels, known as aquaporins (AQPs) has recently been implicated in the regulation of cell motility, and here we provide novel evidence for the role of AQP9 in the development of various forms of membrane protrusion. Using multiple imaging techniques and cellular models we show that: (i) AQP9 induced and accumulated in filopodia, (ii) AQP9-associated filopodial extensions preceded actin polymerization, which was in turn crucial for their stability and dynamics, and (iii) minute, local reductions in osmolarity immediately initiated small dynamic bleb-like protrusions, the size of which correlated with the reduction in osmotic pressure. Based on this, we present a model for AQP9-induced membrane protrusion, where the interplay of water fluxes through AQP9 and actin dynamics regulate the cellular protrusive and motile activity of cells.

  • 56.
    Karlsson, Thommie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Glogauer, Michael
    University of Toronto.
    Ellen, Richard P
    University of Toronto.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magalhaes, Marco A O
    University of Toronto.
    Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization2011In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 90, no 5, p. 963-973Article in journal (Refereed)
    Abstract [en]

    Neutrophils are of prime importance in the host innate defense against invading microorganisms by using two primary mechanisms-locomotion toward and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as AQPs in cell motility. Here, we focused on the role of AQP9 in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of fMLFR or PMA stimulation in primary human neutrophils. The dynamics of AQP9 were assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of WT or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. WT AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway.

  • 57.
    Karlsson, Thommie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lagerholm, Christoffer B.
    University of So Denmark, Denmark .
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Water fluxes through aquaporin-9 prime epithelial cells for rapid wound healing2013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 430, no 3, p. 993-998Article in journal (Refereed)
    Abstract [en]

    Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known about the impact on migrating epithelial sheets during wound healing and epithelial renewal. Here, we investigate and compare the effects of AQP9 on single cell and epithelial sheet migration. To achieve this, MDCK-1 cells stably expressing AQP9 were subjected to migration assessment. We found that AQP9 facilitated cell locomotion at both the single and multi-cellular level. Furthermore, we identified major differences in the monolayer integrity and cell size upon expression of AQP9 during epithelial sheet migration, indicating a rapid volume-regulatory mechanism. We suggest a novel mechanism for epithelial wound healing based on AQP-induced swelling and expansion of the monolayer.

  • 58.
    Karlsson, Thommie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Musse, Farah
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling2012In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 91, no 1, p. 15-26Article in journal (Refereed)
    Abstract [en]

    In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLC gamma 1 but not -gamma 2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.

  • 59.
    Karlsson, Thommie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Turkina, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Yakymenko, Olena
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration2012In: PLOS PATHOGENS, ISSN 1553-7374, Vol. 8, no 10Article in journal (Refereed)
    Abstract [en]

    Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxododecanoyl- L-homoserine lactone 3O-C-12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C-12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C-12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C-12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C-12-HSL with a sensor bioassay. Moreover, 3O-C-12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P. aeruginosa 3O-C-12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C-12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial - human cell communication.

  • 60.
    Keita, Åsa
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine.
    Salim, Sa´ad
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery .
    Jiang, T.
    Department of Clinical and Experimental Medicine Linköping University.
    Yang, P-C
    Intestinal Disease Research Program McMaster University, Hamilton, Canada.
    Franzén, Lennart
    Aleris Medilab Täby.
    Söderkvist, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Söderholm, Johan D
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Surgery . Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Increased uptake of non-pathogenic E. coli via the follicle-associated epithelium in longstanding ileal Crohn's disease2008In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 215, no 2, p. 135-144Article in journal (Refereed)
    Abstract [en]

    In Crohn's disease (CD), inflammation is driven by luminal commensal micro-organisms, however, mechanisms of early phases of inflammation need further clarification. The earliest observable lesions of recurrent CD are microscopic erosions at the specialized follicle-associated epithelium (FAE), which lines the Peyer's patches. Therefore, our aim was to investigate the mucosal barrier to non-pathogenic bacteria in FAE of CD. The FAE of macroscopically normal ileum from patients with longstanding CD, ulcerative colitis, and controls was studied in Ussing chambers regarding electrophysiology and permeability to 51Cr-EDTA, horseradish peroxidase, and non-pathogenic E. coli strains. Transepithelial passage routes and uptake into dendritic cells were studied by confocal and electron microscopy. FAE of CD showed increased numbers of adherent bacteria, after E. coli exposure in Ussing chambers, as well as spontaneously in non-exposed archival surgical tissues. Further, we found increased uptake of fluorescent E. coli K-12 and HB101 across FAE of CD, but not in ulcerative colitis. Microscopy demonstrated intercellular and transcellular uptake of E. coli in CD, but only transcellular in controls. FAE exposed to E. coli demonstrated changes in conductance and 51Cr-EDTA permeability, suggesting that bacteria affected the paracellular pathway in CD mucosa. Following bacterial uptake, CD mucosa also demonstrated an increased percentage of E. coli co-localizing with dendritic cells, and augmented tissue release of TNF-α. Our data present novel insights into the pathophysiology of CD by demonstrating a previously unrecognized defect of FAE barrier to bacteria in ileal CD, leading to increased load of commensal bacteria to the inductive sites of mucosal immunity. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  • 61.
    Khotin, M.G.
    et al.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Turoverova, L.V.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Podolskaya, E.P.
    Institute for Analytical Instrumentation RAS, St. Petersburg, Russian Federation.
    Krasnov, I.A.
    Institute for Analytical Instrumentation RAS, St. Petersburg, Russian Federation.
    Solovyeva, A.V.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Aksenova, V.Yu.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Pinaev, G.P.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Tentler, D.G.
    Institute of Cytology RAS, St. Petersburg, Russian Federation.
    Analysis of nuclear protein complexes comprising a-actinin-4 by 2D-electrophoresis and mass-spectrometry2009In: Tsitologiya, ISSN 0041-3771, Vol. 51, no 8, p. 684-690Article in journal (Refereed)
    Abstract [en]

    Actin-binding protein a-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of a-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with a-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear a-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. ß-Actin, a- and ß-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with ß-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with a-actinin-4 may suggest that a-actinin-4 is involved in transcription and splicing. Presence of a-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDITOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against a-tubulin confirmed association of a-actinin-4 with a-tubulin in the protein complex. Nuclear a-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with a-tubulin. These data suggest that a-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.

  • 62.
    Khotin, Mikhail
    et al.
    Russian Acadamy of Science.
    Turoverova, Lidia
    Russian Acadamy of Science.
    Aksenova, Vasilisa
    Russian Acadamy of Science.
    Barlev, Nikolai
    Russian Acadamy of Science.
    Borutinskaité, Veronika
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology . Linköping University, Faculty of Health Sciences.
    Vener, Alexander
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Bajenova, Olga
    St Petersburg State University.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Pinaev, George P.
    Russian Acadamy of Science.
    Tentler, Dmitri
    Russian Acadamy of Science.
    Proteomic analysis of ACTN4-interacting proteins reveals its a putative involvement in mRNA metabolism2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 397, no 2, p. 192-196Article in journal (Refereed)
    Abstract [en]

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  • 63. Komaraiah, P.
    et al.
    Kishor PB, Kavi
    Carlsson, Maria
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biotechnology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Mandenius, Carl-Fredrik
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Biotechnology .
    Enhancement of anthraquinone accumulation in Morinda citrifolia suspension cultures2005In: Plant Science, ISSN 0168-9452, E-ISSN 1873-2259, Vol. 168, no 5, p. 1337-1344Article in journal (Refereed)
    Abstract [en]

    Enhancement of accumulation of anthraquinones in Morinda citrifolia (Noni fruit) suspension cultures was accomplished by treatment with elicitors, by ultrasonication and by controlled feeding of the carbon source in the growth medium. The elicitation was attained by additions of polyunsaturated fatty acids (linoleic acid, α-linolenic acid, arachidonic acid), methyl jasmonate, salicylate and nitric oxide (by addition of sodium nitroprusside to the medium) at different concentrations. The accumulation of anthraquinones in the elicited cultures ranged from 5 to 12 mg/g dry weight of cells, which was two to three-fold of what was attained in control cultures. Treatment by short pulses of ultrasonication enhanced the accumulation up to 2.5-fold after 16 s of sonication. A synergistic effect was achieved by simultaneously applying elicitation and controlled addition of sucrose, that increased the anthraquinone production to 16.74 mg/g of dry weight, which was more than a four-fold increase above the control cultures. The accumulation of the anthraquinones in the M. citrifolia cells was confirmed by confocal laser fluorescence microscopy. Minor fluorescence was observed from the cells in the lag phase (1-3 days), while substantially higher fluorescence was observed at late exponential and stationary phase (10-14 days). Rounder spherical cells emitted less fluorescence than elongated slender cells. The fluorescence was assumed to be a result of autofluorescent properties of the aromatic anthraquinone molecules. © 2005 Elsevier Ireland Ltd. All rights reserved.

  • 64.
    Kulytè, Agnè
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Navakauskiene, R.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Treigyte, G.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Gineitis, A.
    Institute of Biochemistry, Vilnius, Lithuanuia.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins2001In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 31, no 3, p. 508-517Article in journal (Refereed)
    Abstract [en]

    Phosphotyrosine signaling plays a vital role in cell regulation - from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphorylated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.

  • 65.
    Kulytè, Agnè
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Navakauskiene, Ruta
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Treigyte, Grazina
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Gineitis, Arunas
    Department of Biological Chemistry, School of Medicine, University of California at Davis, Davis, California.
    Bergman, Tomas
    Protein Analysis Center, Karolinska Institutet, Stockholm, Sweden .
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Characterization of Human Alpha-Dystrobrevin Isoforms in HL-60 Human Promyelocytic Leukemia Cells Undergoing Granulocytic Differentiation2002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 12, p. 4195-4205Article in journal (Refereed)
    Abstract [en]

    The biochemical properties and spatial localization of the protein alpha-dystrobrevin and other isoforms were investigated in cells of the human promyelocytic leukemia line HL-60 granulocytic differentiation as induced by retinoic acid (RA). Alpha-dystrobrevin was detected both in the cytosol and the nuclei of these cells, and a short isoform (gamma-dystrobrevin) was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of alpha-dystrobrevin and its isoforms could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and human neutrophils. Moreover, the gamma-dystrobrevin isoform was found in association with actin and myosin light chain. The results provide new information about potential involvement of alpha-dystrobrevin and its splice isoforms in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells.

  • 66.
    Kwak, Young-Keun
    et al.
    Karolinska Institute.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Vecsey-Semjen, Beatrix
    AlbaNova University of Centre.
    Colque-Navarro, Patricia
    Karolinska Institute.
    Mollby, Roland
    Karolinska Institute.
    The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity2012In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 80, no 5, p. 1670-1680Article in journal (Refereed)
    Abstract [en]

    Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca2+](i)), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood.

  • 67.
    Lee, Juliet
    et al.
    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, USA.
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Jacobson, Ken
    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, USA.
    The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes1990In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 247, no 4947, p. 1229-1233Article in journal (Refereed)
    Abstract [en]

    The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence microscopy and digital image-processing techniques were used to analyze a series of images taken at short intervals after photobleaching. The bleached line remained visible for about 5 seconds before being erased by diffusional recovery. Examination of fluorescence intensity profiles allowed a comparison to be made between the velocities of line and cell movement. Results indicate that the bleached line moves forward with the same velocity as the cell during locomotion, refuting the retrograde lipid flow model of locomotion. Instead, the plasma membrane lipid appears to move forward according to either the unit movement of membrane or the tank track model of locomotion.

  • 68.
    Lerm, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holm, Åsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Seiron, Å.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Descoteaux, A.
    INRS- Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
    Rasmusson, Birgitta
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycanManuscript (preprint) (Other academic)
    Abstract [en]

    The intracellular parasite Leishmania donovani survives inside macrophage phagosomes by inhibiting phagosornal maturation. Its main surface glycoconjugate, lipophosphoglycan (LPG), is crucial for survival and essential for the build-up of a coat of F-actin surrounding the phagosome. Previous studies have shown that inhibition of PKCα by LPG is partly responsible for the elevated levels of F-actin around the phagosome (1, 2). This study shows that simultaneous inhibition of Cdc42 and Rac1, members of the Rho family of small GTPases, prevented the accumulation of F-actin around L. donovani containing phagosomes in murine macrophages. Moreover, an LPG-defective L. donovani mutant normally not capable of accumulating F-actin around it's phagosome, displayed elevated amounts of periphagosomal F-actin in cells pre-treated with permanently active forms of Cdc42 and Rac. The lysosomal marker LAMP1 did not translocate normally to phagosomes in these cells, indicating defective phagosomal maturation. We conclude that Cdc42 and Rac are activated by L. donovani in an LPG-dependent manner, and that this activation contributes to the accumulation of periphagosomal F-actin around L. donovani phagosomes. Our results also indicate a direct link between the build-up of periphagosomal F-actinand inhibition of phagosomal mahuation.

  • 69.
    Lerm, Maria
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Holm, Åsa
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Seiron, Å
    Särndahl, Eva
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Rasmusson, Birgitta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation2006In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, no 5, p. 2613-2618Article in journal (Refereed)
    Abstract [en]

    Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase Cα. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

  • 70.
    Lirvall, Margareta
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ljungquist-Höddelius, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts1996In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 16, no 3, p. 227-238Article in journal (Refereed)
    Abstract [en]

    Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz, with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2 ± 0.2 x 10-10 cm2/s, and 1.8 ± 0.2 x 10-10 cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3 ± 0.3 x 10-10 cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1 ± 0.8 x 10-10 cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.

  • 71.
    Lirvall, Margareta
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Majeed, Meytam
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Höddelius, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Rosdahl, Inger
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    UVB radiation increases EGF receptor mobility and trafficking in human melanocytesManuscript (preprint) (Other academic)
    Abstract [en]

    For the human skin, UVB-radiation (290-320nm) is a very potent injurious agent. UV radiation is absorbed in the epidermis and reaching the melanocytes leads to proliferation via activation of growth factor reccptors. This may play a key role in the clonal expansion of melanocytes and be a critical step in carcinogenesis. We show that UVB-irradiated human epidermal melanocytes display an increased mobility of epidermal growth factor receptors (EGF-R) in the plane of the cell membrane, and that UVB affects the intracellular EGF-R transport to the nucleus. Using fluorescence photobleaching technique we show a time and dose dependent increase in the diffusion coefficient and mobile fraction of EGF-R. EGF-Rdiffuse with a low rate within the cell membrane in control cells, and the mobility increases 4-fold after single physiologic doses of UVB. Three-dimensional confocal microscopy reveals that EGF-R display a strilting difference in receptor distribution and intracellular transport before and after UVB irradiation. The EGF-Rclearly eo-localize with clathrin-coated pits within the cells. These results indicate that already single physiologic doses of UVB affect growth factor receptor mobility in cell membranes and intracellular trafficlting. This may be an important early step in the ultraviolet radiation-induced signal transduction pathway leading to cell proliferation.

  • 72.
    Ljungquist-Höddelius, Pia
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lirvall, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lateral diffusion of PDGF β-receptors in human fibroblasts1991In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 11, no 1, p. 43-52Article in journal (Refereed)
    Abstract [en]

    When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum of PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding 'normal' and 'starved' cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10-10 cm2s-1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor. and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

  • 73.
    Loitto, Vesa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Karlsson, Thommie
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Water Flux in Cell Motility: Expanding the Mechanisms of Membrane Protrusion2009In: CELL MOTILITY AND THE CYTOSKELETON, ISSN 0886-1544, Vol. 66, no 5, p. 237-247Article, review/survey (Refereed)
    Abstract [en]

    Transmembrane water fluxes through aquaporins (AQPs) are suggested to play, pivotal roles in cell polarization and directional cell motility. Local dilution by W water influences the dynamics of the subcortical actin polymerization and directs the formation of nascent membrane protrusions. In this paper. recent evidence is discussed in support of such a central role of AQP in membrane protrusion formation, and cell migration as a basis for our Understanding AQP9 Underlying molecular mechanisms of directional motility. Specifically. AQP9 in a physiological context controls transmembrane water fluxes driving, membrane protrusion formation, as an initial cellular response to a chemoattractant or other migratory signals. The importance of AQP-facilitated water fluxes in directional cell motility is underscored the observation that blocking or modifying specific sites in AQP9 also interferes with the molecular machinery that govern actin-mediated cellular shape changes. Cell Motil.

  • 74.
    Loitto, Vesa
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Dysregulation of aquaporins impairs neutrophil leukocyte motility2003In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 84, no 2, p. 519A-519AConference paper (Other academic)
  • 75.
    Loitto, Vesa
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Hg2+ and small-sized polyethylene glycols have inverse effects on membrane permeability, while both impair neutrophil cell motility2004In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 316, no 2, p. 370-378Article in journal (Refereed)
    Abstract [en]

    Toxic effects after exposure to mercury are well documented in human. Little is, however, known about how Hg2+ affect host defense in general and neutrophil functions in particular. We show here that exposure of human neutrophils to HgCl2 dose-dependently impairs chemoattractant-stimulated motility. Long-term exposure (5-10min) to Hg 2+ yields a rapid influx of extracellular Ca2+ followed by leakage of cytosolic fluorophores, as assessed using fura-2 and ratio imaging microscopy. The inhibition on motility was partly reversible, since pre-treated neutrophils placed in an Hg2+-free environment displayed higher migration rates. The Hg2+-induced fluxes were prevented by addition of small-sized polyethylene glycols (PEG 200-400), which also dose-dependently inhibited neutrophil transmigration. Localized, minute micropipette additions of Hg2+ or PEG caused retraction of the leading edge and redirection of cell migration. Since Hg2+ increases and PEGs decrease membrane permeability in a partially competitive manner, we suggest that the known aquaporin-inhibitor Hg2+ alters membrane permeability by affecting the bidirectional flux through the leukocyte aquaporin-9 (AQP9) while small-sized PEGs yield decreased membrane permeability by becoming trapped in the promiscuous channel. The local additions of Hg 2+ or PEG probably force other cell regions to take over from those with blocked AQPs. Hence, the cells turn direction of motility away from the micromanipulator needle.

  • 76.
    Loitto, Vesa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nitric oxide induces dose-dependent changes in [Ca2+](i), morphology and migration of human neutrophils. in MOLECULAR BIOLOGY OF THE CELL, vol 9, issue , pp 290A-290A1998In: MOLECULAR BIOLOGY OF THE CELL, American Society for Cell Biology , 1998, Vol. 9, p. 290A-290AConference paper (Refereed)
    Abstract [en]

    n/a

  • 77.
    Loitto, Vesa-Matti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Forslund, Tony
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Gustafsson, Mikael
    Linköping University, Department of Medicine and Care. Linköping University, Faculty of Health Sciences.
    Neutrophil leukocyte motility requires directed water influx2002In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 71, no 2, p. 212-222Article in journal (Refereed)
    Abstract [en]

    The ability of neutrophils to sense and move to sites of infection is essential for our defense against pathogens. For motility, lamellipodium extension and stabilization are prerequisites, but how cells form such membrane protrusions is still obscure. Using contrast-enhanced video microscopy and Transwell® assays, we show that water-selective aquaporin channels regulate lamellipodium formation and neutrophil motility. Addition of anti-aquaporin-9 antibodies, HgCl2, or tetraethyl ammonium inhibited the function(s) of the channels and blocked motility-related shape changes. On human neutrophils, aquaporin-9 preferentially localized to the cell edges, where N-formyl peptide receptors also accumulated, as assessed with fluorescence microscopy. To directly visualize water fluxes at cell edges, cells were loaded with high dilution-sensitive, self-quenching concentrations of fluorophore. In these cells, motile regions always displayed increased fluorescence compared with perinuclear regions. Our observations provide the first experimental support for motility models where water fluxes play a pivotal role in cell-volume increases accompanying membrane extensions.

  • 78.
    Loitto, Vesa-Matti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    The spatial distribution of RhoA, Rac2 and Cdc42 in human neutrophils allows for sequential chemoattractant stimulation2001In: FEBS Letters, ISSN 0014-5793Article in journal (Refereed)
  • 79.
    Loitto, Vesa-Matti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nilsson, Harriet
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Nitric oxide induces dose-dependent CA2+ transients and causes temporal morphological hyperpolarization in human neutrophils2000In: Journal of cellular physiology, ISSN 0021-9541, Vol. 182, no 3, p. 402-413Article in journal (Refereed)
    Abstract [en]

    We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.

  • 80.
    Loitto, Vesa-Matti
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Rasmusson, Birgitta
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Assessment of neutrophil N-formyl peptide receptors by using antibodies and fluorescent peptides2001In: Journal of Leukocyte biology, ISSN 0741-5400, Vol. 69, no 5, p. 762-771Article in journal (Refereed)
    Abstract [en]

    Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.

  • 81. Loitto, VM
    et al.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Reversible Hg2+ toxicity increases Ca2+ and inhibits neutrophil cell motility2001In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 12, p. 230-Conference paper (Other academic)
  • 82.
    Lutgendorff, Femke
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Nijmeijer, Rian M
    Utrecht University Medical Center.
    Sandström, Per A
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Trulsson, Lena M
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Timmerman, Harro M
    Utrecht University Medical Center.
    van Minnen, L Paul
    Utrecht University Medical Center.
    Rijkers, Ger T
    Utrecht University Medical Center.
    Gooszen, Hein G
    Utrecht University Medical Center.
    Akkermans, Louis M A
    Utrecht University Medical Center.
    Söderholm, Johan D
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Probiotics prevent intestinal barrier dysfunction in acute pancreatitis in rats via induction of ileal mucosal glutathione biosynthesis.2009In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 2, p. e4512-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: During acute pancreatitis (AP), oxidative stress contributes to intestinal barrier failure. We studied actions of multispecies probiotics on barrier dysfunction and oxidative stress in experimental AP. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-three male Spraque-Dawley rats were randomly allocated into five groups: 1) controls, non-operated, 2) sham-operated, 3) AP, 4) AP and probiotics and 5) AP and placebo. AP was induced by intraductal glycodeoxycholate infusion and intravenous cerulein (6 h). Daily probiotics or placebo were administered intragastrically, starting five days prior to AP. After cerulein infusion, ileal mucosa was collected for measurements of E. coli K12 and (51)Cr-EDTA passage in Ussing chambers. Tight junction proteins were investigated by confocal immunofluorescence imaging. Ileal mucosal apoptosis, lipid peroxidation, and glutathione levels were determined and glutamate-cysteine-ligase activity and expression were quantified. AP-induced barrier dysfunction was characterized by epithelial cell apoptosis and alterations of tight junction proteins (i.e. disruption of occludin and claudin-1 and up-regulation of claudin-2) and correlated with lipid peroxidation (r>0.8). Probiotic pre-treatment diminished the AP-induced increase in E. coli passage (probiotics 57.4+/-33.5 vs. placebo 223.7+/-93.7 a.u.; P<0.001), (51)Cr-EDTA flux (16.7+/-10.1 vs. 32.1+/-10.0 cm/s10(-6); P<0.005), apoptosis, lipid peroxidation (0.42+/-0.13 vs. 1.62+/-0.53 pmol MDA/mg protein; P<0.001), and prevented tight junction protein disruption. AP-induced decline in glutathione was not only prevented (14.33+/-1.47 vs. 8.82+/-1.30 nmol/mg protein, P<0.001), but probiotics even increased mucosal glutathione compared with sham rats (14.33+/-1.47 vs. 10.70+/-1.74 nmol/mg protein, P<0.001). Glutamate-cysteine-ligase activity, which is rate-limiting in glutathione biosynthesis, was enhanced in probiotic pre-treated animals (probiotics 2.88+/-1.21 vs. placebo 1.94+/-0.55 nmol/min/mg protein; P<0.05) coinciding with an increase in mRNA expression of glutamate-cysteine-ligase catalytic (GCLc) and modifier (GCLm) subunits. CONCLUSIONS: Probiotic pre-treatment diminished AP-induced intestinal barrier dysfunction and prevented oxidative stress via mechanisms mainly involving mucosal glutathione biosynthesis.

  • 83.
    Magnusson, Karl-Eric
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gustafsson, Mikael
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Cardiology in Linköping.
    Holmgren, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Johansson, Birgitta
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA Class 1, HLA Class 2, and neoplastic epithelial antigens) in the apical cell membrane1990In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 143, no 2, p. 381-390Article in journal (Refereed)
    Abstract [en]

    We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic calcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subuni)-labelled ganglioside GM1 (diffusion coefficient, D [x 108] = 0.8–0.9 cm2s−1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 109] = 2 cm2s−1; R = 60–70%). However, antibody-labelled β2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was × 1.4 and R × 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were × 0.60 and × 0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobil-ity of different membrane components.

  • 84.
    Mahdavi, Jafar
    et al.
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Sondén, Berit
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Hurtig, Martina
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Olfat, Farzad O.
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden and The Swedish Institute for Infectious Disease Control, Solna, Sweden.
    Forsberg, Lina
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Roche, Niamh
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Ångström, Jonas
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Larsson, Thomas
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Teneberg, Susann
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Karlsson, Karl-Anders
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Altraja, Siiri
    Institute of Molecular and Cell Biology, Tartu University, Estonia.
    Wadström, Torkel
    Department of Infectious Diseases and Medical Microbiology, Lund University, Lund, Sweden.
    Kersulyte, Dangeruta
    Department of Molecular Microbiology, Washington University Medical School, St. Louis, USA.
    Berg, Douglas E.
    Department of Molecular Microbiology, Washington University Medical School, St. Louis, USA.
    Dubois, Andre
    Laboratory of Gastrointestinal and Liver Studies, Department of Medicine, USUHS, Bethesda, USA.
    Petersson, Christoffer
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Norberg, Thomas
    Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Lindh, Frank
    IsoSep AB, Tullinge, Sweden.
    Lundskog, Bertil B.
    Department of Medical Biosciences/Clinical Cytology, Umeå University, Umeå, Sweden.
    Arnqvist, Anna
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden and Department of Molecular Biology, Umeå University, Umeå, Sweden.
    Hammarström, Lennart
    Center for Biotechnology, Karolinska Institute, Novum, Huddinge, Sweden.
    Borén, Thomas
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Helicobacter pylori sabA adhesin in persistent infection and chronic inflammation2002In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 297, no 5581, p. 573-578Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori adherence in the human gastric mucosa involves specific bacterial adhesins and cognate host receptors. Here, we identify sialyl-dimeric-Lewis x glycosphingolipid as a receptor for H. pylori and show thatH. pylori infection induced formation of sialyl-Lewis x antigens in gastric epithelium in humans and in a Rhesus monkey. The corresponding sialic acid–binding adhesin (SabA) was isolated with the “retagging” method, and the underlyingsabA gene (JHP662/HP0725) was identified. The ability of many H. pylori strains to adhere to sialylated glycoconjugates expressed during chronic inflammation might thus contribute to virulence and the extraordinary chronicity of H. pylori infection.

  • 85.
    Mahdavi, Jafar
    et al.
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Ögren, Johan
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Ilver, Dag
    Institute of Medical Biochemistry, Göteborg University, Göteborg, Sweden.
    Sondén, Berit
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Christoffer, Petersson
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Odenbreit, Stefan
    Max-von-Pettenkofer of Hygiene and Medical Microbiology, Department of Bacteriology, Munich, Germany.
    Haas, Rainer
    Max-von-Pettenkofer of Hygiene and Medical Microbiology, Department of Bacteriology, Munich, Germany.
    Borén, Thomas
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    Arnqvist, Anna
    Department of Odontology/Oral Microbiology, Umeå University, Umeå, Sweden.
    The blood group antigen binding activity of the Helicobacter pylori baba adhesin is regulated by local ph and redox potentialManuscript (preprint) (Other academic)
    Abstract [en]

    The blood group antigen hinding adhesin, BabA, which binds to fucosylated blood group antigens, such as the Lewis b (Leb) and H1 antigens constitutes one of the best recognized adhesin-receptor interactions that mediate adherence of Helicobacter pylori to the gastric epithelium. BabA belongs to a family of H. pylori outer membrane, proteins (HOPs), a group of some 30 proteins with most similar N- and C-terminal domains.

    We previously identified the babA1 and babA2 genes, where babA2 was found to encode the BabA adhesin in strain CCUG17875. Here, we confirmed the identity of the BabA protein by immunoblot-analysis, followed by MALDIT-OF MS analysis, which also provided molecular weight of the BabA polypeptide and the unique peptide sequences for BabA. Surprisingly, the BabA protein was found to be expressed 50-fold higher compared to the number of calculated bacterial Leb-binding sites.

    Furthermore, surface scan of the bacterial membrane by freeze fracture immuno-EM technique localized the BabA by immunogold labeling to the  bacterial surface in numbers similar to the predicted binding sites. To help explain the binding results, crosslinker-analyses were performed which revealed that BabA form supra-molecular complexes on the bacterial surfaces. In addition, binding to the Lewis b antigen was shown to be pH dependent and took place over a broad pH range but binding activity was reversibly lost when approaching pH 3, i.e. conditions similar to the acidic gastric juice.

    The binding activity of the BabA adhesin was shown to be sensitive for reducing conditions, which suggests the presence of disulfide bond(s) close to the carbohydrate-binding domain. The dynamics of BabA in Leb-binding suggest that the bacterial adhesin is regulated by local variations in pH and redox potential, such as the pH gradient in the slimy mucus lining of the epithelium, and the reduced conditions of the inflamed gastric mucosa.

  • 86.
    Merzvinskyte, Rasa
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine.
    Treigyte, Grazina
    Savickiene, Jurate
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Navakauskiene, Ruta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Effects of histone deacetylase inhibitors, sodium phenyl butyrate and vitamin B3, in combination with retinoic acid on granulocytic differentiation of human promyelocytic leukemia HL-60 cells2006In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1091, p. 356-367Article in journal (Refereed)
    Abstract [en]

    Water-soluble vitamin B3, niacin, and its related compounds were suggested to be applicable for medical use. In this article, we examined the anti-leukemic effects of two distinct histone deacetylase (HDACI and Sir2) inhibitors, sodium phenyl butyrate (PB) and vitamin B3, respectively, on human promyelocytic leukemia cells HL-60, using HDACIs alone and in combination with all trans retinoic acid (RA). We demonstrated that the HDACI combinations exert different effects on cell cycle arrest and differentiation as determined by nitro blue reduction and the expression of the early myeloid differentiation marker CD11b. The most beneficial effects were found by use of 6-h pretreatment with PB and vitamin B3 before the exposition to RA alone or in combination with vitamin B3, showing significant acceleration and a high level of granulocytic differentiation. The effects were associated with a rapid histone 114 acetylation and later histone H3 modifications. Our results suggest that the use of two HDACI altogether before the induction of differentiation and acting via chromatin remodeling may be promising for the treatment of acute promyelocytic leukemia.

  • 87.
    Mirazimi, Ali
    et al.
    Department of Virology, Swedish Institute for Infectious Disease Control/Karolinska Institute, Solna, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Svensson, Lennart
    Linköping University, Department of Molecular and Clinical Medicine, Molecular Virology. Linköping University, Faculty of Health Sciences.
    A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retention in the endoplasmic reticulum2003In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 84, no 4, p. 875-883Article in journal (Refereed)
    Abstract [en]

    The rotavirus genome encodes two glycoproteins, one structural (VP7) and one non-structural (NSP4), both of which mature and remain in the endoplasmic reticulum (ER). While three amino acids in the N terminus have been proposed to function as a retention signal for VP7, no information is yet available on how NSP4 remains associated with the ER. In this study, we have investigated the ER retention motif of NSP4 by producing various C-terminal truncations. Deleting the C terminus by 52 amino acids did not change the intracellular distribution of NSP4, but an additional deletion of 38 amino acids diminished the ER retention and resulted in the expression of NSP4 on the cell surface. Brefeldin A treatment prevented NSP4 from reaching the cell surface, suggesting that C-terminal truncated plasma membrane NSP4 is transported through the normal secretory pathway. On the basis of these results, we propose that the region between amino acids 85 and 123 in the cytoplasmic region of NSP4 are involved in ER retention.

  • 88.
    Molinas, Andrea
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Mirazimi, Ali
    Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.
    Holm, Angelika
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Loitto, Vesa M.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Vikström, Elena
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Protective role of host aquaporin 6 against Hazara virus, a model for Crimean–Congo hemorrhagic fever virus infection2016In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 363, no 8, article id fnw058Article in journal (Refereed)
    Abstract [en]

    Crimean–Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus–host interactions, which may lead to less severe outcomes of an infection.

  • 89.
    Munch, Andreas
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Endocrinology and Gastroenterology UHL.
    Söderholm, Johan D
    Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
    Ost, A
    Medilab, Taby, Sweden .
    Carlsson, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Ström, Magnus
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Endocrinology and Gastroenterology UHL.
    Low levels of bile acids increase bacterial uptake in colonic biopsies from patients with collagenous colitis in remission2011In: ALIMENTARY PHARMACOLOGY and THERAPEUTICS, ISSN 0269-2813, Vol. 33, no 8, p. 954-960Article in journal (Refereed)
    Abstract [en]

    Pandgt;Background Patients with collagenous colitis have an impaired mucosal barrier. Moreover, collagenous colitis is associated with bile acid malabsorption. Bile acids can increase bacterial mucosal uptake in humans. Mucosal barrier function was investigated by exposing colonic biopsies to chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) in Ussing chamber experiments. Aim To find if low levels of bile acids increase bacterial uptake in colonic biopsies from collagenous colitis patients. Methods The study comprised 33 individuals; 25 with collagenous colitis (14 in clinical remission without treatment, 11 with active disease and 10 examined in clinical remission resulting from treatment with 6 mg budesonide); eight healthy individuals undergoing screening colonoscopy served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short-circuit current (Isc), potential difference, trans-epithelial resistance and transmucosal passage of Escherichia coli K12 after adding 100 mu mol/L CDCA or DCA. Results When adding 100 mu mol/L CDCA or DCA, bacterial uptake increased fourfold in biopsies of patients in remission; CDCA 6.5 units [2.5-9.8] and DCA 6.2 units [2.1-22] (median [IQR]), compared with uptake in biopsies without added bile acids 1.6 units [1.1-3] (P = 0.004 and P = 0.01 respectively). In active disease and in patients in remission due to budesonide treatment, bile acids did not affect bacterial uptake. Confocal microscopy revealed trans-epithelial passage of E. coli K12 within 30 min. Conclusions Low concentrations of dihydroxy-bile acids exacerbate mucosal barrier dysfunction in colonic biopsies of patients with collagenous colitis in remission. This allows a substantially increased bacterial uptake, which may contribute to recurrence of inflammation.

  • 90.
    Münch, Andreas
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology . Linköping University, Faculty of Health Sciences.
    Söderholm, Johan
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology.
    Carlsson, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
    Öst, Åke
    Medilab, Täby, Sweden.
    Ström, Magnus
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland.
    Physiological levels of bile acids increase bacterial uptake in colonic biopsies of collagenous colitis patients in remissionManuscript (preprint) (Other academic)
    Abstract [en]

    Objective: Patients with collagenous colitis (CC) have an impaired mucosal barrier. Moreover CC is associated with bile acid malabsorption. Bile acids may increase bacterial mucosal uptake in humans. To elucidate the possible role of bile acids in CC pathophysiology, the mucosal barrier function was investigated by exposing colonic biopsies to physiological concentrations of chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) in Ussing chamber experiments.

    Patients/Interventions: The study included 33 individuals; 25 with collagenous colitis (14 in clinical remission without treatment, 11 with active disease, and 8 of these again after 6 weeks budesonide treatment); 8 healthy individuals undergoing screening colonoscopy served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short circuit current (Isc), transepithelial resistance (TER), and transmucosal passage of chemically killed E. coli K12 after addition of 100 μmol/l CDCA or DCA. The biopsies were further investigated with confocal microscopy to asses bacterial transepithelial passage routes.

    Results: By adding 100μmol/l CDCA or DCA the bacterial uptake was increased by 4-fold in biopsies of patients in remission; CDCA 6.5 units [2.5-9.8] and DCA 6.2 units [2.1-22] (median [IQR]), compared with uptake in biopsies without added bile acids 1.6 units [1.1-3]; (p=0.004 and p=0.01, respectively). In active disease and in patients in remission on budesonide, bile acids had no effect on bacterial uptake. Isc and TER were unaffected by the bile acids at 100μmol/l in all groups. Confocal microscopy demonstrated transepithelial passage of E.coli K12 via the paracellular route.

    Conclusions: Physiological concentrations of dihydroxy-bile acids augment mucosal barrier dysfunction in colonic biopsies of patients with CC in remission. This leads to a substantially increased bacterial uptake that may contribute to relapse of inflammation. Budesonide seems to counteract the bile acid-induced mucosal impairment.

  • 91.
    Navakauskiene, R.
    et al.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania, Department of Developmental Biology, Institute of Biochemistry, Mokslininku 12, LT-08662 Vilnius, Lithuania.
    Treigyte, G.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Gineitis, A.
    Department of Developmental Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Identification of apoptotic tyrosine-phosphorylated proteins after etoposide or retinoic acid treatment of HL-60 cells2004In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 4, no 4, p. 1029-1041Article in journal (Refereed)
    Abstract [en]

    A main shortcoming of using HL-60 cells as a model of granulocyte-macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine-phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL-60 cells were induced specifically to undergo apoptosis with 68 µM etoposide, and to undergo granulocytic differentiation with 1 µM retinoic acid (RA). The corresponding two-dimensional electrophoretic maps of tyrosine-phosphorylated proteins from treated cells were compared. In the 8 h etoposide-treated HL-60 cell population, 83% of the cells were apoptotic. In the 120 h RA-treated cells, 50% of the cells were apoptotic. Eighteen cytosolic and nuclear tyrosine-phosphorylated proteins were found in both the 8 h etoposide- and the 120 h RA-treated cells, but not in the proliferating HL-60 cell population. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry analyses suggested that some of the proteins may be involved in signal transduction pathways (NF?B, GTP-binding protein, protein disulfide isomerase, Cyclophilin A), others in cell transcriptional and translational control (hnRNP H, hnRNP L, Hsp60, Hp1, Hcc-1, 26S proteasome beta-subunit, ATP synthase beta-chain), and a third group in cell cytoskeleton organization and receptor cycling (profilin, caveolin-1). An understanding of signal transduction in apoptosis initiation by screening for tyrosine-phosphorylated proteins associated with apoptosis may provide new targets for the treatment of leukemia.

  • 92.
    Navakauskiene, Ruta
    et al.
    Vilnius University, Lithuania Vilnius Gediminas Technical University, Lithuania .
    Borutinskaite, Veronika V.
    Vilnius University, Lithuania .
    Treigyte, Grazina
    Vilnius University, Lithuania .
    Savickiene, Jurate
    Vilnius University, Lithuania .
    Matuzevicius, Dalius
    Vilnius University, Lithuania Vilnius Gediminas Technical University, Lithuania .
    Navakauskas, Dalius
    Vilnius Gediminas Technical University, Lithuania .
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Epigenetic changes during hematopoietic cell granulocytic differentiation - comparative analysis of primary CD34+cells, KG1 myeloid cells and mature neutrophils2014In: BMC Cell Biology, ISSN 1471-2121, E-ISSN 1471-2121, Vol. 15, no 4Article in journal (Refereed)
    Abstract [en]

    Background: Epigenetic regulation is known to affect gene expression, and recent research shows that aberrant DNA methylation patterning and histone modifications may play a role in leukemogenesis. In order to highlight the co-operation of epigenetic mechanisms acting during the latter process it is important to clarify their potential as biomarkers of granulocytic differentiation. Results: In this study we investigated epigenetic alterations in human hematopoietic cells at a distinct differentiation stages: primary hematopoietic CD34+ cells, KG1 myeloid leukemic cells, whose development is stopped at early stage of differentiation, and mature neutrophils. We focused on the epigenetic status of cell cycle regulating (p15, p16) and differentiation related (E-cadherin and RAR beta) genes. We found that the methylation level in promoter regions of some of these genes was considerably higher in KG1 cells and lower in CD34+ cells and human neutrophils. As examined and evaluated by computer-assisted methods, histone H3 and H4 modifications, i.e. H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc, were similar in CD34+ cells and human mature neutrophils. By contrast, in the KG1 cells, histone H3 and H4 modifications were quite high and increased after induction of granulocytic differentiation with the HDAC inhibitor phenyl butyrate. Conclusions: We found the methylation status of the examined gene promoters and histone modifications to be characteristically associated with the hematopoietic cell progenitor state, induced to differentiate myeloid KG1 cells and normal blood neutrophils. This could be achieved through epigenetic regulation of E-cadherin, p15, p16 and RAR beta genes expression caused by DNA methylation/demethylation, core and linker histones distribution in stem hematopoietic cells, induced to differentiation KG1 cells and mature human neutrophils, as well as the histone modifications H3K4Me3, H3K9Ac, H3K9Ac/S10Ph and H4 hyperAc in relation to hematopoietic cell differentiation to granulocyte. These findings also suggest them as potentially important biomarkers of hematopoietic cell granulocytic differentiation and could be valuable for leukemia induced differentiation therapy.

  • 93.
    Navakauskiene, Ruta
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Kulyte, Agné
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Treigyte, Grazina
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gineitis, Arunas
    Department of Developmental Biology, Institute of Biochemistry, Lithuania, and Department of Biological Chemistry, School of Medicine, University of California at Davis, U.S.A..
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Translocation of transcription regulators into the nucleus during granulocyte commitment of HL-60 cells2003In: Biochemistry and Cell Biology, ISSN 0829-8211, E-ISSN 1208-6002, Vol. 81, no 4, p. 285-295Article in journal (Refereed)
    Abstract [en]

    Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPβ and c-Myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid. c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPβ, which suggests a combinatorial interaction of these transcription factors in the granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation, whereas no significant changes were seen in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincided with augmentation of the STAT5a protein level, which could be evidence of their possible cooperation during granulocytic-lineage commitment of HL-60 cells. Our results suggest that the studied transcription factors cooperatively promote signalling in the differentiating promyelocytic HL-60 cell line in response to retinoic acid.

  • 94.
    Navakauskiene, Ruta
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Kulyté, Agné
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Treigyte, Grazina
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Gineitis, Arunas
    Laboratory of Development Biology, Institute of Biochemistry, Vilnius, Lithuania.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Assessment of transcription regulators and translocation of these proteins into the nucleus during granulocyte lineage commitment of haematopoietis HL-60 cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Expression of transcription factors required for lineage commitment of differentiating cells (C/EBPß and c-myb) and for survival of differentiated cells (STATs and NFκB) was examined in the HL-60 cell line. Differentiation was induced by treating the cells with retinoic acid (RA). c-Myb expression in the nucleus restored at the precommitment stage (18 h) what concurred with the highest nuclear level of C/EBPß, which suggests a combinatorial interaction of these transcription factors in granulocytic signalling pathway. Expression of STAT5a and STAT5b varied during differentiation; there were no significant changes in STAT3 levels. Increased cytosolic level of NFκB p65 during precommitment and commitment stages of granulocytic differentiation coincides with augmentation of STAT5a protein level what could be an evidence of their possible co-operation during granulocyticlineage commitment of HL-60 cells. Our results suggest that the studied proteins cooperatively promote signalling in differentiating promyelocytic HL-60 cell line in response to retinoic acid.

  • 95.
    Navakauskiene, Ruta
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Treigyte, G
    Gineitis, A
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Assessment of p16, p21, and p27 in granulocytic differentiation of human promyelocytic HL-60 cell line2002In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 973, p. 284-286Article in journal (Refereed)
    Abstract [en]

    [No abstract available]

  • 96.
    Navakauskiene, Ruta
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Treigyte, G
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Kulyté, A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Savickiene, J
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Proteomic analysis by MALDI-TOF mass spectrometry and its application to HL-60 cells.2003In: Biologija, ISSN 1392-0146, E-ISSN 2029-0578, Vol. 3, p. 63-65Article in journal (Refereed)
  • 97.
    Navakauskiene, Ruta
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Treigyte, G
    Institute of Biochemistry, LT-08662 Vilnius.
    Savickiene, Jurate
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine.
    Gineitis, A
    Institute of Biochemistry, LT-08662 Vilnius.
    Magnusson, Karl-Eric
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Alterations in protein expression in HL-60 cells during etoposide-induced apoptosis modulated by the caspase inhibitor ZVAD.fmk2004In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1030, p. 393-402Article in journal (Refereed)
    Abstract [en]

    DNA topoisomerase inhibitors induce a specific signaling cascade that promotes an active apoptotic caspase-dependent cell death process. However, little is known about the initial signals elicited by these agents. In the present study, we compared apoptosis in HL-60 cells treated either with the chemotherapeutic drug etoposide (VP16) alone or combined with the broad caspase inhibitor ZVAD.fmk. Apoptosis was assessed by changes in cell morphology and agarose gel electrophoresis of extracted cell DNA. We found that ZVAD.fmk prevents VP16-induced DNA fragmentation and the appearance of an increased number of apoptotic cells in the culture. We also compared the effects of etoposide alone or together with the pan-caspase inhibitor ZVAD.fmk on proliferating cell nuclear antigen, Bcl-2, and actin expression in human promyelocytic leukemia HL-60 cells. In addition, we screened for proteins that were initially upregulated in a caspase-dependent manner. Indeed, some proteins were induced in the cytoplasm and subsequently accumulated in the nuclei after etoposide treatment. This process was slightly inhibited by the caspase inhibitor ZVAD.fmk. We suggest that these proteins are associated with the induction of specific signaling cascades that characterize the apoptotic cell death process.

  • 98.
    Navakauskiene, Ruta
    et al.
    Vilnius University, Lithuania Vilnius Gediminas Technical University, Lithuania .
    Treigyte, Grazina
    Vilnius University, Lithuania .
    Borutinskaite, Veronika-Viktorija
    Vilnius University, Lithuania .
    Matuzevicius, Dalius
    Vilnius Gediminas Technical University, Lithuania .
    Navakauskas, Dalius
    Vilnius Gediminas Technical University, Lithuania .
    Magnusson, Karl-Eric
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Alpha-Dystrobrevin and its associated proteins in human promyelocytic leukemia cells induced to apoptosis2012In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 11, p. 3291-3303Article in journal (Refereed)
    Abstract [en]

    Dystrobrevin is a dystrophin-related component of the dystrophin-associated protein complex (DAPC). Using alpha-dystrobrevin as indicator, we aimed to elucidate the interaction network of the DAPC with other proteins during apoptosis of promyelocytic HL-60 cells. The precise role(s) of DBs are not known, but we and others have shown that they play a role in intracellular signal transduction and cellular organization. Apoptosis was induced with etoposide in the absence or presence of Z-VAD to block caspase activity, and we then followed the cellular distribution of alpha-DB and its association with other proteins, using confocal imaging and cell fractions analyses after immune-precipitation with anti-alpha-DB and mass spectrometry. Confocal imaging revealed distinct spatial relocalizations of alpha-DB between the cell membrane, cytosol and nucleus after induction of apoptosis. The expression levels of the identified proteins were evaluated with computer-assisted image analysis of the gels. We thus identified associations with structural and transport proteins (tropomyosin, myosin), membrane (ADAM21, syntrophin), ER-Golgi (TGN51, eIF38) and nuclear (Lamins, ribonucleoprotein C1/C2) proteins. These results suggest that apoptosis-induction in HL-60 cells involves not only classical markers of apoptosis but also a network alpha-DB-associated proteins at the cell membrane, the cytoplasm and nucleus, affecting key cellular transport processes and cellular structure.

  • 99.
    Petersson, Christoffer
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Forsberg, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Aspholm, Marina
    Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
    Olfat, Farzad O.
    The Swedish Institute for Infectious Disease Control, Solna, Sweden and Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
    Forslund, Tony
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Borén, Thomas
    Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Helicobacter pylori sabA adhesin evokes a strong inflammatory response in human neutrophils which is down-regulated by the neutrophil-activating protein2006In: Medical Microbiology and Immmunology, ISSN 0300-8584, E-ISSN 1432-1831, Vol. 195, no 4, p. 195-206Article in journal (Refereed)
    Abstract [en]

    The human pathogen Helicobacter pylori expresses two dominant adhesins; the Lewis b blood group antigen binding adhesin, BabA, and the sialic acid-binding adhesin, SabA. These adhesins recognize specific carbohydrate moieties of the gastric epithelium, i.e. the Lewis b antigen, Leb, and the sialyl-Lewis x antigen, sLex, respectively, which promote infection and inflammatory processes in the gastroduodenal tract. To assess the contribution of each of BabA, SabA and the neutrophil activating protein (HP-NAP) in a local inflammation, we investigated the traits of H. pylori mutants in their capacity to interact with and stimulate human neutrophils. We thence found that the SabA adhesin was not only the key inducer of oxidative metabolism (Unemo et al. J Biol Chem 280:15390–15397, 2005), but also essential in phagocytosis induction, as evaluated by flow cytometry, fluorescence microscopy and luminol-enhanced chemiluminescence. The napA deletion resulted in enhanced generation of reactive oxygen species and impaired adherence to the host cells. In conclusion, the SabA adhesin stimulates human neutrophils through selectin-mimicry. Interestingly, HP-NAP modulates the oxidative burst, which could tune the impact of the H. pylori infection for establishment of balanced and chronic inflammation of the gastric mucosa.

  • 100.
    Petersson, Christoffer
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Larsson, Bertil
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Mahdavi, Jafar
    Department of Odontology, Umeå University, Umeå, Sweden .
    Borén, Thomas
    Department of Odontology, Umeå University, Umeå, Sweden .
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    A new method to visualize the helicobacter pylori-associated lewisb-binding adhesin utilizing SDS-digested freeze-fracture replica labeling2000In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 48, no 6, p. 877-883Article in journal (Refereed)
    Abstract [en]

    Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.

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