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  • 1.
    Andersson, C
    et al.
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden.
    Söderström, M
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden.
    Mannervik, B
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden.
    Activation and inhibition of microsomal glutathione transferase from mouse liver.1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 249, no 3, p. 819-23Article in journal (Refereed)
    Abstract [en]

    Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.

  • 2. Antunes, Fernando
    et al.
    Cadenas, Enrique
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Apoptosis, induced by exposure to a low and steady-state concentration of H2O2, is a consequence of lysosomal rupture2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 356, p. 549-555Article in journal (Refereed)
  • 3. Baird, Sarah K
    et al.
    Kurz, Tino
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Metallothionein protects against oxidative stress-induced lysosomal destabilization2006In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 394, no 1, p. 275-283Article in journal (Refereed)
    Abstract [en]

    The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. © 2006 Biochemical Society.

  • 4.
    Berndt, Carsten
    et al.
    Division for Biochemistry, Department for Medical Biochemistry and Biophysics, Karolinska Institute, Sweden. Institute for Cinical Cytobiology and Cytopathology, Philipps-Universität, Marburg, Germany .
    Kurz, Tino
    Linköping University, Department of Medicine and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Selenius, Markus
    Division of Pathology, Department of Laboratory Medicineand Division of Medical Radiation Physics, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.
    Fernandes, Aristi P
    Division of Pathology, Department of Laboratory Medicineand Division of Medical Radiation Physics, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.
    Edgren, Margareta R
    Division of Medical Radiation Physics, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.
    Brunk, Ulf T
    Linköping University, Department of Medicine and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Chelation of lysosomal iron protects against ionizing radiation.2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 432, no 2, p. 295-301Article in journal (Refereed)
    Abstract [en]

    Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.

  • 5.
    Dunlop, Rachael A
    et al.
    Cell Biology Group, Heart Research Institute, Newton, NSW 2042, Australia.
    Brunk, Ulf T
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Rodgers, Kenneth J
    Cell Biology Group, Heart Research Institute, Newton, NSW 2042, Australia.
    Proteins containing oxidized amino acids induce apoptosis in human monocytes.2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 435, no 1, p. 207-216Article in journal (Refereed)
    Abstract [en]

    Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.

  • 6.
    Favre, Cécile J.
    et al.
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Jerström, Petra
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Foti, Michelangelo
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Huggler, Elzbieta
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Lew, Daniel P.
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Krause, Karl-Heinz
    Division of Infectious Diseases, University Hospital, Geneva, Switzerland.
    Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor1996In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 316, no 1, p. 137-142Article in journal (Refereed)
    Abstract [en]

    In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.

  • 7.
    Fälker, Knut
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry.
    Haglund, Linda
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Nylander, Martina
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Chemistry.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 436, p. 469-480Article in journal (Refereed)
    Abstract [en]

    FARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(alpha 12/13) and G(alpha q) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca(2+) mobilization, PKC (protein kinase C) signalling and alpha-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y(12) receptor-induced G(alpha i) signalling accounted for the loss of the aggregation response, as mimicking G(alpha i/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from alpha-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

  • 8.
    Jufvas, Åsa
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Rajan, Meenu Rohini
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Jönsson, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Turkina, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences.
    Scaffolding protein IQGAP1: an insulin-dependent link between caveolae and the cytoskeleton in primary human adipocytes?2016In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 473, no 19, p. 3177-3188Article in journal (Refereed)
    Abstract [en]

    The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by five-fold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.

  • 9.
    Karlsson, Markus
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
    Kurz, Tino
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Brunk, Ulf T.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Nilsson, Sven E.
    Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
    Frennesson, Christina I.
    Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences.
    What does the commonly used DCF test for oxidative stress really show?2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 428, no 2, p. 183-90Article in journal (Refereed)
    Abstract [en]

    H(2)DCF-DA (dihydrodichlorofluorescein diacetate) is widely used to evaluate 'cellular oxidative stress'. After passing through the plasma membrane, this lipophilic and non-fluorescent compound is de-esterified to a hydrophilic alcohol [H(2)DCF (dihydrodichlorofluorescein)] that may be oxidized to fluorescent DCF (2',7'-dichlorofluorescein) by a process usually considered to involve ROS (reactive oxygen species). It is, however, not always recognized that, being a hydrophilic molecule, H(2)DCF does not cross membranes, except for the outer fenestrated mitochondrial ones. It is also not generally realized that oxidation of H(2)DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor H(2)O(2), directly oxidizes H(2)DCF. Consequently, oxidation of H(2)DCF requires the presence of either cytochrome c or of both redox-active transition metals and H(2)O(2). Redox-active metals exist mainly within lysosomes, whereas cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H(2)DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to H(2)O(2), showing that by itself it is unable to oxidize H(2)DCF. Cells that were either exposed to the lysosomotropic detergent MSDH (O-methylserine dodecylamide hydrochloride), exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

  • 10.
    Kurz, Tino
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Leake, Alan
    von Zglinicki, Thomas
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 378, no 3, p. 1039-1045Article in journal (Refereed)
    Abstract [en]

    Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243-1252, Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295-1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O 2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride, subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.

  • 11.
    Kågedal, Katarina
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Zhao, Ming
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Svensson, Irene
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Brunk, Ulf
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Sphingosine-induced apoptosis is dependent on lysosomal proteases2001In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 359, no 2, p. 335-343Article in journal (Refereed)
    Abstract [en]

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.

  • 12.
    Li, Wei
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences.
    Yuan, Ximing
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine.
    Ivanova, S.
    Laboratory of Biomedical Science, North Shore-LIE Research Institute, Manhasset, NY 11030, United States.
    Tracey, K.J.
    Laboratory of Biomedical Science, North Shore-LIE Research Institute, Manhasset, NY 11030, United States.
    Eaton, John Wallace
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Pharmacology .
    3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 371, no 2, p. 429-436Article in journal (Refereed)
    Abstract [en]

    Cytotoxic polyamine-derived amino aldehydes, formed during cerebral ischaemia, damage adjacent tissue (the so-called 'penumbra') not subject to the initial ischaemic insult. One such product is 3-aminopropanal (3-AP), a potent cytotoxin that accumulates in ischaemic brain, although the precise mechanisms responsible for its formation are still unclear. More relevant to the present investigations, the mechanisms by which such a small aldehydic compound might be cytotoxic are also not known, but we hypothesized that 3-AP, having the structure of a weak lysosomotropic base, might concentrate within lysosomes, making these organelles a probable focus of initial toxicity. Indeed, 3-AP leads to lysosomal rupture of D384 glioma cells, a process which clearly precedes caspase activation and apoptotic cell death. Immunohistochemistry reveals that 3-AP concentrates in the lysosomal compartment and prevention of this accumulation by the lysosomotropic base ammonia, NH3, protects against 3-AP cytotoxicity by increasing lysosomal pH. A thiol compound, N-(2-mercaptopropionyl)glycine, reacts with and neutralizes 3-AP and significantly inhibits cytoxocity. Both amino and aldehyde functions of 3-AP are necessary for toxicity: the amino group confers lysosomotropism and the aldehyde is important for additional, presently unknown, reactions. We conclude that 3-AP exerts its toxic effects by accumulating intralysosomally, causing rupture of these organelles and releasing lysosomal enzymes which initiate caspase activation and apoptosis (or necrosis if the lysosomal rupture is extensive). These results may have implications for the development of new therapeutics designed to lessen secondary damage arising from focal cerebral ischaemia.

  • 13.
    Los, Marek Jan
    et al.
    Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany..
    Droge, W.
    Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany..
    Schulze-Osthoff, Klaus
    Division of Immunochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany..
    Inhibition of Activation of Transcription Factor Ap-1 by Cd28 Signaling1994In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 302, p. 119-123Article in journal (Refereed)
    Abstract [en]

    Co-stimulation of T-lymphocytes by T-cell receptor (TcR) occupancy and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production. The increase in IL-2 gene expression triggered by CD28 involves a KB-like sequence in the 5'-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12-myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-B-K. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-1. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity of NF-B-K, PMA-induced activation of AP-1 was significantly suppressed. The inhibitory effect exerted by anti-CD38 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation.

  • 14.
    Miranda-Vizuete, A.
    et al.
    Novum, Karolinska Institutet, Huddinge, Sweden.
    Spyrou, Giannis
    Novum, Karolinska Institutet, Huddinge, Sweden.
    The novel oxidoreductase KDRF (KM-102-derived reductase-like factor) is identical with human thioredoxin reductase1997In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 325 ( Pt 1), p. 287-288Article in journal (Refereed)
  • 15.
    Neuzil, Jiri
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Zhao, Ming
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Ostermann, Georg
    Sticha, Martin
    Gellert, Nina
    Weber, Christian
    Eaton, John Wallace
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Alfa-Tocopheryl succinate, an agent with in vivo anti-tumour activity, induces apoptosis by causing lysosomal instability2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 362, p. 709-715Article in journal (Refereed)
  • 16.
    Persson, H.Lennart
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Respiratory Medicine.
    Kurz, Tino
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Pharmacology .
    Eaton, John Wallace
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Pharmacology .
    Radiation-induced cell death: Importance of lysosomal destabilization2005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 389, no 3, p. 877-884Article in journal (Refereed)
    Abstract [en]

    The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. In addition to the direct formation of highly reactive hydroxyl radicals (HO.) by radiolysis of water, oxidative stress events in the cytoplasm due to formation of H2O2 may also be important. Since the major pool of low-mass redox-active intracellular iron seems to reside within lysosomes, arising from the continuous intralysosomal autophagocytotic degradation of ferruginous materials, formation of H2O2 inside and outside these organelles may cause lysosomal labilization with release to the cytosol of lytic enzymes and low-mass iron. If of limited magnitude, such release may induce 'reparative autophagocytosis', causing additional accumulation of redox-active iron within the lysosomal compartment. We have used radio-resistant histiocytic lymphoma (J774) cells to assess the importance of intralysosomal iron and lysosomal rupture in radiation-induced cellular injury. We found that a 40 Gy radiation dose increased the 'loose' iron content of the (still viable) cells approx. 5-fold when assayed 24 h later. Cytochemical staining revealed that most redox-active iron was within the lysosomes. The increase of intralysosomal iron was associated with 'reparative autophagocytosis', and sensitized cells to Iysosomal rupture and consequent apoptotic/necrotic death following a second, much lower dose of radiation (20 Gy) 24 h after the first one. A high-molecular-mass derivative of desferrioxamine, which specifically localizes intralysosomally following endocytic uptake, added to the culture medium before either the first or the second dose of radiation, stabilized lysosomes and largely prevented cell death. These observations may provide a biological rationale for fractionated radiation. © 2005 Biochemical Society.

  • 17.
    Psarra, Anna-Maria G
    et al.
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Hermann, Stefan
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Panayotou, George
    Biomedical Sciences Research Center Alexander Fleming, Laboratory of Protein Chemistry, Vari, Greece.
    Spyrou, Giannis
    Biomedical Research Foundation, Academy of Athens, Center of Basic Research, Athens, Greece.
    Interaction of mitochondrial thioredoxin with glucocorticoid receptor and NF-κB modulates glucocorticoid receptor and NF-κB signalling in HEK-293 cells2009In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 422, no 3, p. 521-531Article in journal (Refereed)
    Abstract [en]

    Trx2 (mitochondrial thioredoxin) is an antioxidant and anti-apoptotic factor essential for cell viability. Trx1 (cytoplasmic thioredoxin) is a co-factor and regulator of redox-sensitive transcription factors such as the GR (glucocorticoid receptor) and NF-kappaB (nuclear factor kappaB). Both transcription factors have been detected in mitochondria and a role in mitochondrial transcription regulation and apoptosis has been proposed. In the present study, we show using SPR (surface plasmon resonance) and immunoprecepitation that GR and the p65 subunit of NF-kappaB are Trx2-interacting proteins. The interaction of Trx2 with GR is independent of the presence of GR ligand and of redox conditions. The p65 subunit of NF-kappaB can interact with Trx2 in the oxidized, but not the reduced, form. Using HEK (human embryonic kidney)-293 cell lines with increased or decreased expression of Trx2, we show that Trx2 modulates transcription of GR and NF-kappaB reporter genes. Moreover, Trx2 overexpression modulates the mRNA levels of the COX1 (cytochrome oxidase subunit I) and Cytb (cytochrome b), which are known to be regulated by GR and NF-kappaB. Increased expression of Trx2 differentially affects the expression of Cytb. The glucocorticoid dexamethasone potentiates the expression of Cytb, whereas TNFalpha (tumour necrosis factor alpha) down-regulates it. These results suggest a regulatory role for Trx2 in GR and NF-kappaB signalling pathways.

  • 18. Quinn, Carmel M.
    et al.
    Kågedal, Katarina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Terman, Alexei
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Stroikin, Yuri
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion, Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Jessup, Wendy
    Garner, B
    Induction of fibroblast apolipoprotein E expression during apoptosis, starvation-induced growth arrest and mitosis2004In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 378, no 3, p. 753-761Article in journal (Refereed)
    Abstract [en]

    Apolipoprotein E (apoE) mediates the hepatic clearance of plasma lipoproteins, facilitates cholesterol efflux from macrophages and aids neuronal lipid transport. ApoE is expressed at high levels in hepatocytes, macrophages and astrocytes. In the present study, we identify nuclear and cytosolic pools of apoE in human fibroblasts. Fibroblast apoE mRNA and protein levels were up-regulated during staurosporine-induced apoptosis and this was correlated with increased caspase-3 activity and apoptotic morphological alterations. Because the transcription of apoE and specific pro-apoptotic genes is regulated by the nuclear receptor LXR (liver X receptor) α, we analysed LXRα mRNA expression by quantitative real-time PCR and found it to be increased before apoE mRNA induction. The expression of ABCA1 (ATP-binding cassette transporter A1) mRNA, which is also regulated by LXRα, was increased in parallel with apoE mRNA, indicating that LXRα probably promotes apoE and ABCA1 transcription during apoptosis. Fibroblast apoE levels were increased under conditions of serum-starvation-induced growth arrest and hyperoxia-induced senescence. In both cases, an increased nuclear apoE level was observed, particularly in cells that accumulated lipofuscin. Nuclear apoE was translocated to the cytosol when mitotic nuclear disassembly occurred and this was associated with an increase in total cellular apoE levels. ApoE amino acid sequence analysis indicated several potential sites for phosphorylation. In vivo studies, using 32P-labelling and immunoprecipitation, revealed that fibroblast apoE can be phosphorylated. These studies reveal novel associations and potential roles for apoE in fundamental cellular processes.

  • 19.
    Staffas, Louise
    et al.
    Wallenberg Laboratory, Stockholm University, Sweden.
    Mankowitz, Louise
    Wallenberg Laboratory, Stockholm University, Sweden.
    Söderström, Mats
    Wallenberg Laboratory, Stockholm University, Sweden.
    Blanck, Agneta
    Huddinge University Hospital, Sweden.
    Porsch-Hällström, Inger
    Huddinge University Hospital, Sweden.
    Sundberg, Carola
    Wallenberg Laboratory, Stockholm University, Sweden.
    Mannervik, Bengt
    Uppsala University, Sweden.
    Olin, Birgit
    Uppsala University, Sweden.
    Rydström, Jan
    Royal Institute of Technology, Stockholm, Sweden.
    DePierre, Joseph W
    Wallenberg Laboratory, Stockholm University, Sweden.
    Further characterization of hormonal regulation of glutathione transferase in rat liver and adrenal glands. Sex differences and demonstration that growth hormone regulates the hepatic levels1992In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 286 ( Pt 1), p. 65-72Article in journal (Refereed)
    Abstract [en]

    Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutathione transferase subunits could be altered by continuous infusion of growth hormone to both sham-operated and hypophysectomized rats of both sexes. This treatment feminized the isoenzyme pattern in sham-operated males and a similar effect was obtained upon treating hypophysectomized rats with thyroxine, cortisone acetate and a continuous infusion of growth hormone.

  • 20.
    Söderström, Mats
    et al.
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Sweden.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Mannervik, Bengt
    Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Sweden.
    Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 250, no 3, p. 713-718Article in journal (Refereed)
    Abstract [en]

    Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4:glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4.

  • 21. Tenopoulou, M
    et al.
    Doulias, PT
    Barbouti, A
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Galaris, D
    Role of compartmentalized redox-active iron in hydrogen peroxide-induced DNA damage and apoptosis2005In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 387, no 3, p. 703-710Article in journal (Refereed)
    Abstract [en]

    Jurkat cells in culture were exposed to oxidative stress in the form of continuously generated hydrogen peroxide, obtained by the addition of glucose oxidase to the medium. This treatment induced a rapid, dose-dependent increase in the ICIP (intracellular calcein-chelatable iron pool). Early destabilization of lysosomal membranes and subsequent nuclear DNA strand breaks were also observed, as evaluated by the Acridine Orange relocation test and the comet assay respectively. Somewhat later, these effects were followed by a lowered mitochondrial membrane potential, with release of cytochrome c and apoptosis-inducing factor. These events were all prevented if cells were pretreated with the potent iron chelator DFO (desferrioxamine) for a period of time (2-3 h) long enough to allow the drug to reach the lysosomal compartment following fluid-phase endocytosis. The hydrophilic calcein, a cleavage product of calcein acetoxymethyl ester following the action of cytosolic esterases, obviously does not penetrate intact lysosomal membranes, thus explaining why ICIP increased dramatically following lysosomal rupture. The rapid decrease in ICIP after addition of DFO to the medium suggests draining of cytosolic iron to the medium, rather than penetration of DFO through the plasma membrane. Most importantly, these observations directly connect oxidative stress and resultant DNA damage with lysosomal rupture and the release of redox-active iron into the cytosol and, apparently, the nucleus. © 2005 Biochemical Society.

  • 22.
    Tenopoulou, Margarita
    et al.
    Ioannina, Greece.
    Kurz, Tino
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Doulias, Paschalis-Thomas
    Ioannina, Greece.
    Galaris, Dimitrios
    Ioannina, Greece.
    Brunk, Ulf
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?2007In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 403, no 2, p. 261-266Article in journal (Refereed)
    Abstract [en]

    The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at < pH 5. In the present study we show that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron. © 2007 Biochemical Society.

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