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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.
    Appelkvist, Eewa-Liisa
    et al.
    Karolinska Institutet, Huddinge Hospital, Sweden.
    Söderström, Mats
    Arrhenius Laboratory for the Natural Sciences, Stockholm University, Sweden.
    Nässberger, Lennart
    Stockholm University, Sweden.
    Damberg, Charlotta
    Stockholm University, Sweden.
    Dallner, Gustav
    Karolinska Institutet, Huddinge Hospital, Sweden / Stockholm University, Sweden.
    DePierre, Joseph W
    Stockholm University, Sweden.
    Characterization of the lipid and protein contents of myelin bodies isolated from the renal cortex of gentamicin-treated rats1991In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 181, no 2, p. 894-901Article in journal (Refereed)
    Abstract [en]

    Myelin bodies were isolated from the renal cortex of gentamicin-treated rats (100 mg/kg body weight, twice daily for 3 days, i.p.) employing an initial pelleting by differential centrifugation and subsequent flotation on a discontinuous sucrose gradient. These structures were found to contain almost twice as much protein as phospholipid and SDS-polyacrylamide gel electrophoresis revealed the presence of many different polypeptides. All the major phospholipids are present, although myelin bodies contain a considerably higher proportion of phosphatidylinositol, somewhat more phosphatidylcholine and considerably lower percentages of phosphatidylserine and sphingomyelin than do normal renal phospholipids. The fatty acids of myelin body phospholipids are highly saturated (67.3-87.9%) and a striking feature is the occurrence of relatively large amounts of 22:1, presumably erucic acid, especially in sphingomyelin. Myelin bodies contain small amounts of unesterified cholesterol, unesterified dolichol and coenzymes Q9 and Q10.

  • 3.
    Berg, Cecilia
    et al.
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Linköping University, Faculty of Health Sciences.
    Herbertsson, Helena
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Lindström, Eva
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Svensson, Ann-Charlotte
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Division of cell biology. Linköping University, Faculty of Health Sciences.
    Tengvall, Pentti
    Linköping University, The Institute of Technology. Linköping University, Department of Physics, Chemistry and Biology, Applied Physics.
    Bengtsson, Torbjörn
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase2006In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 96, no 5, p. 652-659Article in journal (Refereed)
    Abstract [en]

    Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A2 inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1-2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis. © 2006 Schattauer GmbH, Stuttgart.

  • 4.
    Castro, Victor M
    et al.
    University of Uppsala, Sweden.
    Söderström, Mats
    University of Stockholm, Sweden.
    Carlberg, Inger
    University of Stockholm, Sweden.
    Widersten, Mikael
    University of Uppsala, Sweden.
    Platz, Anton
    University of Stockholm, Sweden.
    Mannervik, Bengt
    University of Uppsala, Sweden.
    Differences among human tumor cell lines in the expression of glutathione transferases and other glutathione-linked enzymes1990In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 11, no 9, p. 1569-1576Article in journal (Refereed)
    Abstract [en]

    A large number of human tumor cell lines of various origins have been investigated with respect to expression of glutathione-linked enzymes in the cytosol fraction. The amounts of the different enzymes were estimated by use of activity measurements and by silver staining or immunoblot analysis after electrophoresis of cytosol fractions purified by affinity chromatography on S-hexylglutathione Sepharose. Class Pi glutathione transferase was the most abundant enzyme in most tumor cells; the cell lines HepG2 and Raji were exceptions in not expressing significant amounts of this enzyme. HepG2 cells derive from hepatocytes, which normally do not express the class Pi enzyme, whereas Raji cells originate from B-lymphocytes, which normally do express a class Pi glutathione transferase. The highest level of the class Pi transferase, in terms of protein reacting with antibodies as well as enzyme activity, was noted in the colon carcinoma cell line LS174T. Hu549Pat cells, EBV-transformed B-lymphocytes, also expressed high levels of a protein reacting with antibodies specific for class Pi glutathione transferases, but did not display any significant activity with ethacrynic acid, a substrate characteristic for this class. Class Alpha and class Mu glutathione transferases, in cell lines expressing these isoenzymes, were present in significantly lower concentrations than the class Pi enzyme. Most of the tumor cells contained a class Alpha transferase composed of 27.5 kd subunits, which has the physicochemical and immunological properties of the most basic glutathione transferase found in human skin. In several cell lines, a protein was detected with an apparent subunit Mr value of 30 kd that was tentatively identified as an additional class Alpha glutathione transferase not previously described. In addition, other glutathione-linked enzyme activities, namely glutathione peroxidase, glutathione reductase and glyoxalase I, were assayed with specific substrates in the cytosolic fraction of the tumor cells; glyoxalase I could also be estimated semiquantitatively by silver staining of SDS-PAGE cells after affinity chromatography. Like the glutathione transferases, these enzymes displayed distinctly different levels of expression in the various cell lines. Thus, virtually every cell line was found to have a unique pattern of glutathione-linked enzymes, suggesting that the resistance phenotypes of the cells differ accordingly.

  • 5.
    Davidsson, Anette
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Physiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart Centre, Department of Clinical Physiology.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Naidu Sjöswärd, Kerstin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Schmekel, Birgitta
    Linköping University, Department of Medical and Health Sciences, Clinical Physiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart Centre, Department of Clinical Physiology.
    Chlorine in Breath Condensate: A Measure of Airway Affection in Pollinosis?2007In: Respiration, ISSN 0025-7931, E-ISSN 1423-0356, Vol. 74, no 2, p. 184-191Article in journal (Refereed)
    Abstract [en]

    Background: Infiltration of inflammatory cells in bronchial mucosa and glandular hypersecretion are hallmarks of asthma. It has been postulated that exhaled breath condensate (EBC) mirrors events in epithelial lining fluid of airways, such as presence of local inflammation as well as glandular hypersecretion. It is also well known that eosinophil cationic protein (ECP) and cysteinyl-leukotrienes (cys-LT) are released by circulating inflammatory cells when triggered by antigen stimulation in asthma patients.

    Objectives: The aim of this study was to evaluate whether chlorine and/or cys-LT in EBC would reflect changes of exposure of airborne pollen in patients with asthma.

    Methods: EBC and serum were collected from 23 patients with allergic asthma during a pollen season and repeated 5 months later during a period with no aeroallergens. Chlorine was measured by means of a sensitive coulometric technique and cys-LT by an EIA technique. Serum ECP was measured and lung function tests were performed and symptoms noted during both occasions.

    Results: Significantly higher concentrations of chlorine in EBC (p = 0.007) and ECP in serum (p = 0.003) were found during the pollen season compared to post-season. Chlorine levels tended to be higher in patients who reported of chest symptoms compared to those who denied symptoms during the pollen season (p = 0.06). Areas under the receiver-operated characteristic curves (AUCROC) were compared and similar discriminative power to identify exacerbations of asthma was recorded by chlorine in EBC (range 0.67-0.78) and ECP in serum (range 0.64-0.78).

    Conclusion: It is concluded that chlorine in EBC and ECP in serum decreased significantly post-season, and this is suggested to mirror the decrement in airborne antigen. It is furthermore proposed that chlorine in EBC and ECP in serum tend to have a similar capacity to identify seasonal variations in airborne pollen in patients with asthma.

  • 6.
    DiRenzo, J
    et al.
    University of California, San Diego, La Jolla, USA.
    Söderström, Mats
    University of California, San Diego, La Jolla, USA.
    Kurokawa, R.
    University of California, San Diego, La Jolla, USA.
    Ogliastro, MH
    University of California, San Diego, La Jolla, USA.
    Ricote, M
    University of California, San Diego, La Jolla, USA.
    Ingrey, S
    University of California, San Diego, La Jolla, USA.
    Hörlein, A
    University of California, San Diego, La Jolla, USA.
    Rosenfeld, M G
    University of California, San Diego, La Jolla, USA.
    Glass, C K
    University of California, San Diego, La Jolla, USA.
    Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors1997In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 17, no 4, p. 2166-2176Article in journal (Refereed)
    Abstract [en]

    As the obligate member of most nuclear receptor heterodimers, retinoid X receptors (RXRs) can potentially perform two functions: cooperative binding to hormone response elements and coordinate regulation of target genes by RXR ligands. In this paper we describe allosteric interactions between RXR and two heterodimeric partners, retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs); RARs and PPARs prevent and permit activation by RXR-specific ligands, respectively. By competing for dimerization with RXR on response elements consisting of direct-repeat half-sites spaced by 1 bp (DR1 elements), the relative abundance of RAR and PPAR determines whether the RXR signaling pathway will be functional. In contrast to RAR, which prevents the binding of RXR ligands and recruits the nuclear receptor corepressor N-CoR, PPAR permits the binding of SRC-1 in response to both RXR and PPAR ligands. Overexpression of SRC-1 markedly potentiates ligand-dependent transcription by PPARgamma, suggesting that SRC-1 serves as a coactivator in vivo. Remarkably, the ability of RAR to both block the binding of ligands to RXR and interact with corepressors requires the CoR box, a structural motif residing in the N-terminal region of the RAR ligand binding domain. Mutations in the CoR box convert RAR from a nonpermissive to a permissive partner of RXR signaling on DR1 elements. We suggest that the differential recruitment of coactivators and corepressors by RAR-RXR and PPAR-RXR heterodimers provides the basis for a transcriptional switch that may be important in controlling complex programs of gene expression, such as adipocyte differentiation.

  • 7.
    Guidez, F
    et al.
    Chester Beatty Laboratories, London, UK.
    Ivins, S
    Chester Beatty Laboratories, London, UK.
    Zhu, J
    Chester Beatty Laboratories, London, UK.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Waxman, S
    Mount Sinai School of Medicine, New York, USA.
    Zelent, A
    Chester Beatty Laboratories, London, UK.
    Reduced retinoic acid-sensitivities of nuclear receptor corepressor binding to PML- and PLZF-RARalpha underlie molecular pathogenesis and treatment of acute promyelocytic leukemia1998In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 91, no 8, p. 2634-2642Article in journal (Refereed)
    Abstract [en]

    Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARalpha fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RARalpha fusion protein. Both PML- and PLZF-RARalpha possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RARalpha protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RARalpha, it had a very little effect on its association with the PLZF-RARalpha fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RARalpha was mediated by the N-terminal PLZF moiety of the chimera. It appears that N-CoR/histone deacetylase corepressor complex interacts directly in an ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF protein and is required, at least in part, for its function as a transcriptional repressor. As the above-noted results predict, histone deacetylase inhibitors antagonize oncogenic activities of the PML-RARalpha fusion protein and partially relieve transcriptional repression by PLZF as well as inhibitory effect of PLZF-RARalpha on ATRA response. Taken together, our results demonstrate involvement of nuclear receptor corepressor/histone deacetylase complex in the molecular pathogenesis of APL and provide an explanation for differential sensitivities of PML- and PLZF-RARalpha-associated leukemias to ATRA.

  • 8.
    Hammarström, Sven
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Trinks, Cecilia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Wigren, Jane
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Surapureddi, S
    Söderström, Mats
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Division of cell biology.
    Glass, CK
    Novel eicosanoid activators of PPAR? formed by raw 264.7 macrophage cultures2002In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 507, p. 343-349Article in journal (Refereed)
    Abstract [en]

    [No abstract available]

  • 9.
    Hammarström, Sven
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Trinks, Cecilia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wigren, Jane
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Surapureddi, Sailesh
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Glass, Christopher K
    University of California, San Diego, Jolla, CA, USA.
    Novel eicosanoid activators of PPAR gamma formed by RAW 264.7 macrophage cultures2002In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 507, p. 343-349Article in journal (Refereed)
  • 10.
    Heinzel, T.
    et al.
    University of California, San Diego, USA.
    Lavinsky, R. M.
    University of California, San Diego, USA.
    Mullen, T. M.
    University of California, San Diego, USA.
    Söderström, Mats
    University of California, San Diego, USA.
    Laherty, C. D.
    Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
    Torchia, J.
    University of California, San Diego, USA.
    Yang, W. M.
    University of South Florida, Tampa, USA.
    Brard, G.
    University of California, San Diego, USA.
    Ngo, S. D.
    University of California, San Diego, USA.
    Davie, J. R.
    University of Manitoba, Winnipeg, Canada.
    Seto, E.
    University of South Florida, Tampa, USA.
    Eisenman, R. N.
    Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
    Rose, D. W.
    University of California, San Diego, USA.
    Glass, C. K.
    University of California, San Diego, USA.
    Rosenfeld, M. G.
    University of California, San Diego, USA.
    A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression1997In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 387, no 6628, p. 43-48Article in journal (Refereed)
    Abstract [en]

    Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.

  • 11.
    Hörlein, A. J.
    et al.
    University of California, San Diego, USA.
    Näär, A. M.
    University of California, San Diego, USA.
    Heinzel, T.
    University of California, San Diego, USA.
    Torchia, J.
    University of California, San Diego, USA.
    Gloss, B.
    University of California, San Diego, USA.
    Kurokawa, R.
    University of California, San Diego, USA.
    Ryan, A.
    University of California, San Diego, USA.
    Kamei, Y.
    University of California, San Diego, USA.
    Söderström, Mats
    University of California, San Diego, USA.
    Glass, C. K.
    University of California, San Diego, USA.
    Rosenfeld, M. G.
    University of California, San Diego, USA.
    Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor1995In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 377, no 6548, p. 397-404Article in journal (Refereed)
    Abstract [en]

    Thyroid-hormone and retinoic-acid receptors exert their regulatory functions by acting as both activators and repressors of gene expression. A nuclear receptor co-repressor (N-CoR) of relative molecular mass 270K has been identified which mediates ligand-independent inhibition of gene transcription by these receptors, suggesting that the molecular mechanisms of repression by thyroid-hormone and retinoic-acid receptors are analogous to the co-repressor-dependent transcriptional inhibitory mechanisms of yeast and Drosophila.

  • 12.
    Kurahashi, Yuko
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Herbertsson, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Rosenfeld, Michael G
    University of California, San Diego, La Jolla, CA, USA.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    A 12(S)-hydroxyeicosatetraenoic acid receptor interacts with steroid receptor coactivator-12000In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 97, no 11, p. 5779-5783Article in journal (Refereed)
    Abstract [en]

    Lewis lung carcinoma cells contain specific high-affinity binding sites for the eicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE]. These binding sites have a cytosolic/nuclear localization and contain the heat shock proteins hsp70 and hsp90 as components of a high molecular weight cytosolic binding complex. The ligand binding subunit of this complex is a protein with an apparent molecular mass of ÿ50 kDa as judged by gel permeation chromatography. In this report, we present data showing that the 50-kDa 12(S)-HETE binding protein interacts as a homodimer with steroid receptor coactivator-1 (SRC-1) in the presence of 12(S)-HETE. Two putative interaction domains were mapped. One of these (amino acids 701-781) was within the nuclear receptor interaction domain in SRC-1 required for binding of various steroid and thyroid hormone receptors. It contains the most C-terminal of the three copies of LXXLL motif present in the nuclear receptor interaction domain. The second interaction domain was present in the N-terminal part of SRC-1 (amino acids 1-221). This region has two LXXLL motifs, one does not bind and the other binds only weakly to steroid and thyroid hormone receptors. Glutathione S-transferase (GST) pulldown experiments and far Western analyses demonstrated that the N-terminal region of SRC-1 (amino acids 1-212) alone does not bind the 50-kDa 12(S)-HETE binding protein, whereas GST/?SRC-11-1138 ligand-dependently pulled down a protein of ÿ50 kDa in size. Our results suggest that the 50-kDa 12(S)-HETE binding protein is a receptor that may signal through interaction with a nuclear receptor coactivator protein.

  • 13.
    Kurokawa, R.
    et al.
    University of California, San Diego, USA.
    Söderström, Mats
    University of California, San Diego, USA.
    Hörlein, A.
    University of California, San Diego, USA.
    Halachmi, S.
    Dana Farber Cancer Institute, Boston, Massachusetts, USA .
    Brown, M.
    Dana Farber Cancer Institute, Boston, Massachusetts, USA .
    Rosenfeld, M. G.
    University of California, San Diego, USA.
    Glass, C. K.
    University of California, San Diego, USA.
    Polarity-specific activities of retinoic acid receptors determined by a co-repressor.1995In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 377, no 6548, p. 451-454Article in journal (Refereed)
    Abstract [en]

    Retinoic acid receptors (RARs) and retinoid-X receptors (RXRs) activate or repress transcription by binding as heterodimers to DNA-response elements that generally consist of two direct repeat half-sites of consensus sequence AGGTCA. On response elements consisting of direct repeats spaced by five base pairs (DR + 5 elements), RAR/RXR heterodimers activate transcription in response to RAR-specific ligands, such as all-trans-retinoic acid (RA). In contrast, on elements consisting of direct repeats spaced by one base pair (DR + 1 elements), RAR/RXR heterodimers exhibit little or no response to activating ligands and repress RXR-dependent transcription. Here we show that ligand-dependent transactivation by RAR on DR + 5 elements requires the dissociation of a new nuclear receptor co-repressor, N-CoR, and recruitment of the putative co-activators p140 and p160. Surprisingly, on DR + 1 elements, N-CoR remains associated with RAR/RXR heterodimers even in the presence of RAR ligands, resulting in constitutive repression. These observations indicate that DNA-response elements can allosterically regulate RAR-co-repressor interactions to determine positive or negative regulation of gene expression.

  • 14.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-β gene2005In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 97, no 4, p. 322-327Article in journal (Refereed)
    Abstract [en]

    Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ). Transient transfection experiments now suggest that TRβ and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-β (TSHβ) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRβ and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRβ cooperate in the regulation of the TSHβ gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.

  • 15.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)2004In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 91, no 4-5, p. 191-196Article in journal (Refereed)
    Abstract [en]

    Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor β and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif.

    This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoRLDNLL) fused to GST interacted with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ) in vitro. Similarly, [35S-methionine]N-CoRLDNLL interacted with a RARα fusion protein. N-CoRLDNLL also bound to RARα in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARα in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARα and TRβ.

  • 16.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    N-CoR interacts directly with the DNA sequence ATNNTNCTCManuscript (preprint) (Other academic)
    Abstract [en]

    N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of thyroid and retinoid hormone receptors) are large proteins which are constituent parts of numerous multicomponent repressor complexes. They function by recruiting repressors to the sites of DNA-bound transcription factors, thereby modifying chromatin condensation to restrict access for general transcription factors to a regulated gene. Both N-CoR and SMRT contain two highly conserved SANT (SWI3, ADA2, N-CoR, TFIIIB) domains which, by homology, have been suggested to confer DNA-binding properties. To date however, there have been no data published that support a DNA-binding function for N-CoR (or SMRT). To investigate if N-CoR is capable of binding to DNA, we used stably transfected S2 cells expressing FLAGtagged N-CoR We then allowed N-CoR to interact with double stranded degenerated oligonucleotides. Our results showed that oligonucleotides were retained by N-CoR, and that these contained the consensus sequence ATNNTNCTC.

  • 17.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    The nuclear receptor corepressor (N-CoR) modulates basal and activated transcription of genes controlled by retinoic acid2003In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 84, no 1, p. 15-21Article in journal (Refereed)
    Abstract [en]

    Variation in cell morphology and function is caused by differentiation. In myeloid differentiation, retinoid signaling, acting through heterodimers consisting of retinoic acid receptor and retinoid X receptor (RAR/RXR) plays a crucial part. The RAR/RXR heterodimers bind to naturally occurring response elements in the promoter regions of target genes, deciding whether the gene is to be transcribed or not. In the absence of the RAR-specific ligand all trans retinoic acid, RAR/RXR heterodimers are associated with the nuclear receptor corepressor N-CoR or the related SMRT.

    Here we show, using Western, far-Western and Northern blot techniques, that when the human monocytic cell line THP-1 is allowed to differentiate into macrophage-like cells the expression of N-CoR is down-regulated both at the protein and at the mRNA level. To investigate how this affects the transcriptional activity of retinoic acid response element (RARE)-controlled genes, we performed transient transfection experiments in THP-1 and CV-1 cells. The results indicate that N-CoR functions not merely as a repressor of basal transcription, but rather as a modulator of both basal and ligand-activated transcription of genes controlled by RAR/RXR heterodimers in a dose-dependent manner.

  • 18.
    Sauma, Lilian
    et al.
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Franck, Niclas
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Paulsson, Johan F
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla T.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kjølhede, Preben
    Linköping University, Department of Molecular and Clinical Medicine. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Nyström, Fredrik H.
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Peroxisome proliferator activated receptor gamma activity is low in mature primary human visceral adipocytes2007In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 50, no 1, p. 195-201Article in journal (Refereed)
    Abstract [en]

    AIMS/HYPOTHESIS: The amount of visceral fat mass strongly relates to insulin resistance in humans. The transcription factor peroxisome proliferator activated receptor gamma (PPARG) is abundant in adipocytes and regulates genes of importance for insulin sensitivity. Our objective was to study PPARG activity in human visceral and subcutaneous adipocytes and to compare this with the most common model for human disease, the mouse.

    MATERIALS AND METHODS: We transfected primary human adipocytes with a plasmid encoding firefly luciferase controlled by PPARG response element (PPRE) from the acyl-CoA-oxidase gene and measured PPRE activity by emission of light. RESULTS: We found that PPRE activity was 6.6-fold higher (median) in adipocytes from subcutaneous than from omental fat from the same subjects (n = 23). The activity was also 6.2-fold higher in subcutaneous than in intra-abdominal fat cells when we used a PPARG ligand-binding domain-GAL4 fusion protein as reporter, demonstrating that the difference in PPRE activity was due to different levels of activity of the PPARG receptor in the two fat depots. Stimulation with 5 micromol/l rosiglitazone did not induce a PPRE activity in visceral adipocytes that was as high as basal levels in subcutaneous adipocytes. Interestingly, in mice of two different strains the PPRE activity was similar in visceral and subcutaneous fat cells.

    CONCLUSIONS/INTERPRETATION: We found considerably lower PPARG activity in visceral than in subcutaneous primary human adipocytes. Further studies of the molecular mechanisms behind this difference could lead to development of drugs that target the adverse effects of visceral obesity.

  • 19.
    Sauma, Lilian
    et al.
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Stenkula, Karin G
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kjølhede, Preben
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Nyström, Fredrik H
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    PPAR-gamma response element activity in intact primary human adipocytes: effects of fatty acids2006In: Nutrition (Burbank, Los Angeles County, Calif.), ISSN 0899-9007, E-ISSN 1873-1244, Vol. 22, no 1, p. 60-68Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: We studied the activity and regulation of the peroxisome proliferator-activated receptor-gamma response element (PPRE) in primary human adipocytes.

    METHODS: We transfected primary human adipocytes with a plasmid-encoding firefly luciferase cDNA under control of a PPRE from the acyl-coenzyme A oxidase gene by using our newly developed electroporation-based method. Several fatty acids were added to the fat cells to study potential activation of peroxisome proliferator-activated receptor-gamma.

    RESULTS: Cells responded maximally to 5 microM of rosiglitazone at a 5.1 +/- 1.4-fold over basal increase in luciferase activity. There was a positive correlation between body mass index and the response to 5 microM of rosiglitazone (r = 0.36, P = 0.03). Patients with type 2 diabetes had similar basal PPRE activity but responded more strongly to 5 microM of rosiglitazone than did non-diabetic subjects (10.2 +/- 5-fold and 5.4 +/- 1-fold over basal increase, respectively, P < 0.0001). Among saturated fatty acids, lauric acid was without effect, but 10 microM of palmitic or stearic acid increased PPRE activity 20% to 35% above basal levels. Monounsaturated palmitoleic acid at 1 microM induced a PPRE transcriptional activity that corresponded to half the therapeutic levels of rosiglitazone.

    CONCLUSION: Adipocytes from obese subjects and patients with type 2 diabetes responded particularly strongly to the effect of rosiglitazone on PPRE. Because fatty acids in the diet can affect the transcriptional activity of peroxisome proliferator-activated receptor-gamma over decades, the stimulation induced by stearic and palmitoleic acids can affect insulin sensitivity and, hence, cardiovascular morbidity and mortality in humans.

  • 20.
    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.

  • 21.
    Stenkula, Karin G.
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Said Suma, Lilian
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Karlsson, Margareta
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Thorn, Hans
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kjölhede, Preben
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center of Paediatrics and Gynaecology and Obstetrics, Department of Gynaecology and Obstetrics in Linköping.
    Gustavsson, Johanna
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Strålfors, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Nyström, Fredrik H
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes2004In: Molecular and Cellular Endocrinology, ISSN 0303-7207, Vol. 221, no 1-2, p. 1-8Article in journal (Refereed)
    Abstract [en]

    Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% ± 5 (mean ± S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

  • 22.
    Strid, Tobias
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Karlsson, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Zhang, Jie
    University of California.
    Qian, Hong
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Sigvardsson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 383, no 3, p. 336-339Article in journal (Refereed)
    Abstract [en]

    Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (540), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence from in situ hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45(-) cells and CD45(+) hematopoietic cells. The latter were further separated into immature (Lin(-)) and mature (Lin(+)) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45(+) cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.

  • 23.
    Strid, Tobias
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Sigvardsson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Karlsson, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Qian, Hong
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Expression of leukotriene biosynthesis proteins in fetal and adult hematopoietic cells and its functional effects on hematopoiesisManuscript (preprint) (Other academic)
    Abstract [en]

    Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5-lipoxygenase activating protein (FLAP) is also required. We have previously reported expression of FLAP in the hematopoietic compartment of the fetal liver raising questions regarding the role of leukotrienes in hematopoietic regulation. Here we report evidence from in situ hybridization, immunohistochemistry and qRT-PCR experiments that the complete LT biosynthesis machinery is abundantly expressed in hematopoietic cells of the fetal mouse liver from e11.5 until birth. FACS sorting of hematopoietic cells from e15.5 liver and adult bone marrow into different subpopulations followed by quantitative RT-PCR analysis showed that expression was confined mainly to myeloid cells but also detected in hematopoietic stem and progenitor cells. Analysis of FLAP knockout mice showed that a lack of this gene abolished LT and reduced 5(S)- hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (HETE) production. Furthermore,  decreased relative numbers of B-lymphocytes and increased numbers of T-lymphocytes were observed in peripheral blood and increased numbers of common lymphoid progenitor cells were observed in BM. Taken together these findings suggest that production of LTs can occur in cells of the fetal and adult hematopoietic compartments and that deficiency of the FLAP gene (and leukotrienes) may affect lymphocyte maturation.

  • 24.
    Strid, Tobias
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Svartz, Jesper
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Franck, Niclas
    Linköping University, Department of Medical and Health Sciences, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Hallin, Elisabeth
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ingelsson, Björn
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Distinct parts of leukotriene C-4 synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein2009In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 381, no 4, p. 518-522Article in journal (Refereed)
    Abstract [en]

    Leukotriene C-4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C-4 synthase (LTC4S) participate in its biosynthesis. We report evidence that LTC4S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC4S. FLAP bound to the N-terminal part/first hydrophobic region of LTC4S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC4S. Fluorescent FLAP- and LTC4S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC4S co-localized with a fluorescent ER marker. In testing HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC4S. Direct interaction of 5-LO and LTC4S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC4S.

  • 25.
    Strid, Tobias
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Cellbiologi IKE.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Leukotriene C4 synthase promoter driven expression of GFP reveals cell specificity2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 366, no 1, p. 80-85Article in journal (Refereed)
    Abstract [en]

    Leukotriene C4 synthase is a key enzyme in leukotriene biosynthesis. Its gene has been cloned and mapped to mouse chromosome 11. Expression occurs in cells of myeloid origin and also in the choroid plexus, the hypothalamus and the medial eminence of mouse brain. In this study a vector that expresses enhanced green fluorescent protein (eGFP) under the control of the mouse leukotriene C4 synthase promoter was constructed and used to study promoter activity in different cell lines. Specific eGFP expression was observed in human monocytic leukemia (THP-1) and rat basophilic leukemia (RBL-1) myeloid cells which both express leukotriene C4 synthase, but not in human embryonic kidney (HEK293/T) epithelial cells which do not express this enzyme. In the myeloid cells, but not in the epithelial cells, we observed that the leukotriene C4 synthase promoter activity was stimulated by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid. In contrast dimethyl sulfoxide did not affect promoter activity. © 2007 Elsevier Inc. All rights reserved.

  • 26.
    Surapureddi, Sailesh
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Morgenstern, Ralf
    Institute of Environmental Medicine, Karolinska Institute, S-171 77, Stockholm, Sweden.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Interaction of human leukotriene C4 synthase and microsomal glutathione transferase in vivo1996In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 229, no 2, p. 388-395Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase (mGT) specifically binds leukotriene C4 synthase in the presence of Mg2+ ion (Söderström et al., Protein Expression and Purification (1995) 6, 352-356). To investigate if this interaction occurs in vivo we screened a human lung cDNA library with a bait vector encoding human mGT in the yeast two-hybrid system. One of the five positive clones obtained encoded leukotriene C4 synthase. This clone was expressed in two heterologous systems. The recombinant protein cross-reacted with a guinea pig antibody raised against a Keyhole limpet hemocyanin coupled synthetic peptide corresponding to amino acids 141-150 of human leukotriene C4 synthase.

  • 27.
    Surapureddi, Sailesh
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Svartz, Jesper
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. 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.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis2000In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 480, no 2-3, p. 239-243Article in journal (Refereed)
    Abstract [en]

    We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC4 production.

  • 28.
    Svartz, Jesper
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Blomgran, Robert
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer2003In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1879-2618, Vol. 1633, no 2, p. 90-95Article in journal (Refereed)
    Abstract [en]

    Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.

  • 29.
    Svartz, Jesper
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hallin, Elisabeth
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Franck, Niclas
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Strid, Tobias
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Studies of interactions between leukotriene C4 synthase, five-lipoxygenase activating protein and 5-lipoxygenaseManuscript (preprint) (Other academic)
    Abstract [en]

    Cysteinyl leukotrienes (cysLTs) are biologically active lipid mediators of great importance in asthma and inflammation. Three proteins are required to convert arachidonic acid into leukotriene C4 namely: five-lipoxygenase (5-LO), five-lipoxygenase activating protein (FLAP) and leukotriene C4 synthase (LTC4S). LTC4S and FLAP belong to the MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) family of proteins and are located on the nuclear envelope. Upon cell activation 5-LO translocates from the cytosol to the nuclear envelope enabling protein-protein interactions to occur between the three biosynthetic enzymes. GST pull-down experiments in this study demonstrate interaction between LTC4S and 5-LO, LTC4S and FLAP and between FLAP and 5-LO. Experiments with truncated mutants indicated that the second hydrophilic loop of LTC4S is important for interaction with 5-LO, and that the N-terminal part of LTC4S is important for FLAP interaction. Bioluminescence resonance energy transfer (BRET) experiments in transfected cells provided additional evidence that LTC4S interacts with 5-LO.

  • 30.
    Svartz, Jesper
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hallin, Elisabeth
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Shi, Yixuan
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization2006In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 98, no 6, p. 1517-1527Article in journal (Refereed)
    Abstract [en]

    Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6–27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60–89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114–135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117–132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix–helix interactions in the membrane.

  • 31.
    Svensson, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet fragments, like platelets, induce airway smooth muscle cell proliferation through mechanisms dependent on ros and 5-lox2007Conference paper (Refereed)
  • 32.
    Svensson Holm, Ann-Charlotte
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Berg, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Herbertsson, Helena
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    5-Lipoxygenase activity is involved in platelet-induced fibroblast proliferation2006Conference paper (Refereed)
  • 33.
    Svensson Holm, Ann-Charlotte
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Berg, Cecilia
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Herbertsson, Helena
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    5-Lipoxygenase activity is involved in platelet-induced fibroblast proliferation2006Conference paper (Refereed)
  • 34.
    Söderberg, Anita
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Barral, Anna-Maria
    Linköping University, Department of Biomedicine and Surgery. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Sander, Birgitta
    Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden.
    Rosén, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Redox-signaling transmitted in trans to neighboring cells by melanoma-derived TNF-containing exosomes2007In: Free Radical Biology and Medicine, ISSN 0891-5849, Vol. 43, no 1, p. 90-99Article in journal (Refereed)
    Abstract [en]

    Hydrogen peroxide is known to be involved in redox signaling pathways that regulate normal processes and disease progression, including cytokine signaling, oxidative stress, and cancer. In studies on immune surveillance against cancer, hydrogen peroxide was found to disrupt cytotoxic T-cell function, thus contributing to tumor escape. In this study, secretion of TNF-containing vesicles of rab9+ endosomal origin, termed exosomes, was investigated using GFP-TNF constructs. We observed a polarized intracellular trafficking and apical secretion of TNF-positive nanovesicles. Cell-to-cell transfer of TNF was observed in exosomes in real-time microscopy, occurring separate from the melanin/melanosome compartment. Exosomes were prepared by ultracentrifugation or immunoisolation on anti-β2-microglobulin magnetic beads. TNF as well as TNF receptors 1 and 2 were present in the exosomes as determined by Western blot, flow cytometry, and deconvolution microscopy. The functional significance of melanoma-derived exosomes was established by their signaling competence with ability to generate significantly higher ROS levels in T cells compared with sham exosomes (P = 0.0006). In conclusion, we report here, for the first time, that TNF is found in tumor cell-derived exosomes and that these exosomes transmit redox signaling in trans to neighboring cells. The results are of importance for a better understanding of tumor escape mechanisms.

  • 35.
    Söderström, M
    et al.
    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.
    Orning, L
    Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden.
    Hammarström, S
    Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden.
    Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase.1985In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 128, no 1, p. 265-70Article in journal (Refereed)
    Abstract [en]

    The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.

  • 36.
    Söderström, Mats
    et al.
    Stockholm University, Sweden.
    Bolling, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Induction of leukotriene C4 synthase activity in differentiating human erythroleukemia cells1992In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 189, no 2, p. 1043-1049Article in journal (Refereed)
    Abstract [en]

    Leukotriene (LT)C4 synthase is a membrane-bound, specific glutathione transferase which catalyzes the transformation of LTA4 to LTC4. It was originally shown to be present in rodent mastocytoma and basophilic leukemia cells as well as in macrophages. Recently, expression of human LTC4 synthase was demonstrated in platelets (Söderström, M., et al. (1992) Arch. Biochem. Biophys. 294, 70-74). The present report describes the induction of LTC4 synthase activity during differentiation of human erythroleukemia (HEL) cells by the protein kinase C stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA), ligands of the steroid-thyroid hormone receptor superfamily: all-trans-retinoic acid (RA) and 1 alpha, 25-dihydroxy-vitamin D3 and in addition dimethylsulfoxide (DMSO). TPA was the most powerful inducer of enzyme activity followed by 1 alpha, 25-dihydroxy-vitamin D3 and DMSO. RA did not induce LTC4 synthase activity.

  • 37.
    Söderström, Mats
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Engblom, David
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Blomqvist, Anders
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Hammarström, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Expression of leukotriene C4 synthase mRNA by the choroid plexus in mouse brain suggests novel neurohormone functions of cysteinyl leukotrienes2003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 307, no 4, p. 987-990Article in journal (Refereed)
    Abstract [en]

    Leukotriene C4 is a potent mediator of allergic and inflammatory reactions, and is formed from arachidonic acid and glutathione through the sequential action of 5-lipoxygenase and leukotriene C4 synthase (LTCS). These enzymes are predominantly expressed in cells of myeloid lineage. In this report, we have investigated LTCS mRNA expression in mouse brain. Expression was demonstrated using RT-PCR and RNase protection assays. In situ hybridization experiments showed exclusive staining of the choroid plexus of all brain ventricles. This expression pattern may provide a mechanism for the generation of LTC4 on the cerebral side of the blood-brain barrier and suggests a possible novel regulator function of LTC4 in the formation of cerebrospinal fluid.

  • 38.
    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.

  • 39.
    Söderström, Mats
    et al.
    Wallenberg Laboratory, Stockholm University, Sweden.
    Mannervik, Bengt
    Uppsala University, Sweden.
    Garkov, Vladimir
    Pennsylvania State University, USA.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    On the nature of leukotriene C4 synthase in human platelets1992In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 294, no 1, p. 70-74Article in journal (Refereed)
    Abstract [en]

    Leukotriene C4 is considered to play a major role in several important pathophysiological conditions, e.g., allergy, asthma, and shock. The present investigation demonstrates the presence in human platelets of a membrane-associated enzyme catalyzing the final step in the biosynthesis of leukotriene C4. This leukotriene C4 synthase was shown to be distinct from previously characterized "microsomal" and soluble glutathione transferases. The latter enzymes did not contribute significantly to the leukotriene A4 conjugating activity in platelets. As determined with leukotriene C4 synthase of a crude membrane fraction from human platelets, the Km value was 7 microM and the V value was 0.56 nmol x min-1 x mg-1 with leukotriene A4 as substrate. The enzyme was 20-fold more efficient with leukotriene A4 than with leukotriene A5 and 30-fold more efficient than with the unphysiological derivative leukotriene A4 methyl ester, as measured by the corresponding V/Km values; 14,15-leukotriene A4 was not a substrate. Platelets should be a useful source for the purification and further characterization of human leukotriene C4 synthase.

  • 40.
    Söderström, Mats
    et al.
    Stockholm University, Sweden.
    Mannervik, Bengt
    Uppsala University, Sweden.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Leukotriene C4 synthase: characterization in mouse mastocytoma cells1990In: Methods in Enzymology, ISSN 0076-6879, E-ISSN 1557-7988, Vol. 187, p. 306-312Article in journal (Refereed)
    Abstract [en]

    The chapter presents a study on leukotriene C4 (LTC4) synthase, discussing the characterization in mouse mastocytoma cells. LTC4 is formed by conjugation of leukotriene A4 (LTA4) with glutathione (GSH). In biological systems, the reaction is catalyzed by a membrane-bound enzyme, leukotriene C4 synthase (EC 2.5.1.37). Cytosolic glutathione transferases, in particular, members of the class Mu, have been shown to catalyze formation of LTC4.The most efficient isoenzymes are transferase 6-6 isolated from rat brain, transferase 4-4 from rat liver, and transferase μ from human liver. The name leukotriene C4 synthase, used for the enzyme described in this chapter, has been adopted to distinguish the enzyme from the above glutathione transferases, which display broad substrate specificity. Reports from three groups of investigators have shown that LTC4 formation in rat basophilic leukemia cells is catalyzed by a membrane-bound enzyme. Leukotriene C4 synthase activity has been described and an enzyme partially purified from the microsomal fraction of guinea pig lung. The formation of LTC4 is especially high in mouse mastocytoma cells, the source from which LTC4 was first isolated. The partial purification of leukotriene C4 synthase from this source is described in the chapter.

  • 41.
    Söderström, Mats
    et al.
    Wallenberg Laboratory, Stockholm University, Sweden.
    Morgenstern, Ralf
    Karolinska Institute, Stockholm, Sweden.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Protein-protein interaction affinity chromatography of leukotriene C4 synthase1995In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 6, no 3, p. 352-356Article in journal (Refereed)
    Abstract [en]

    A novel affinity chromatography purification for human leukotriene C4 synthase is described. It is based on a specific interaction between leukotriene C4 synthase and microsomal glutathione S-transferase which occurs in the presence of magnesium ion. Microsomal glutathione S-transferase was immobilized on NHS-activated Sepharose 4B and used as an affinity matrix. Microsomes from 12-O-tetradecanoyl phorbol 13-acetate-treated human erythroleukemia cells were solubilized with taurocholic acid and applied on the affinity matrix at 0.1 M Mg2+ concentration. After washing with a buffer containing Mg2+, the enzyme was eluted with a glutathione-containing buffer lacking Mg2+. This facile one-step procedure gave a 166-fold purification of leukotriene C4 synthase with a yield of 44%. Analyses of proteins specifically adsorbed to the affinity matrix revealed components with apparent molecular weights of 18, 37, 48, and 60 kDa.

  • 42.
    Söderström, Mats
    et al.
    University of California, San Diego, La Jolla, USA.
    Vo, A
    University of California, San Diego, La Jolla, USA.
    Heinzel, T
    University of California, San Diego, La Jolla, USA.
    Lavinsky, R M
    University of California, San Diego, La Jolla, USA.
    Yang, W M
    H. Lee Moffitt Cancer Center and Research Institute, University of South Florida,Tampa, USA.
    Seto, E
    H. Lee Moffitt Cancer Center and Research Institute, University of South Florida,Tampa, USA.
    Peterson, D A
    Salk Institute for Biology, La Jolla, California, USA.
    Rosenfeld, M G
    University of California, San Diego, La Jolla, USA.
    Glass, C K
    University of California, San Diego, La Jolla, USA.
    Differential effects of nuclear receptor corepressor (N-CoR) expression levels on retinoic acid receptor-mediated repression support the existence of dynamically regulated corepressor complexes1997In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 11, no 6, p. 682-692Article in journal (Refereed)
    Abstract [en]

    Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors that stimulate the transcription of target genes in the presence of activating ligands and repress transcription in their absence. Transcriptional repression by the thyroid hormone and retinoic acid receptors has been proposed to be mediated by the nuclear receptor corepressor, N-CoR, or the related factor, SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). Recent studies have suggested that transcriptional repression by N-CoR involves a corepressor complex that also contains mSin3A/B and the histone deacetylase, RPD3. In this manuscript, we demonstrate that transcriptional repression by the retinoic acid receptor can be either positively or negatively regulated by changes in the levels of N-CoR expression, suggesting a relatively strict stoichiometric relationship between N-CoR and other components of the corepressor complex. Consistent with this interpretation, overexpression of several functionally defined domains of N-CoR also relieve repression by nuclear receptors. N-CoR is distributed throughout the nucleus in a nonuniform pattern, and a subpopulation becomes concentrated into several discrete dot structures when highly expressed. RPD3 is also widely distributed throughout the nucleus in a nonuniform pattern. Simultaneous imaging of RPD3 and N-CoR suggest that a subset of each of these proteins colocalize, consistent with the existence of coactivator complexes containing both proteins. In addition, a substantial fraction of both N-CoR and mSin3 A/B appear to be independently distributed. These observations suggest that interactions between RPD3 and Sin3/N-CoR complexes may be dynamically regulated.

  • 43.
    Söderström, Mats
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wigren, Jane
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Surapureddi, Sailesh
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Glass, Christopher K
    University of California, San Diego, La Jolla, CA , USA.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Novel prostaglandin D2-derived activators of peroxisome proliferator-activated receptor-γ are formed in macrophage cell cultures2003In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1631, no 1, p. 35-41Article in journal (Refereed)
    Abstract [en]

    Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9alpha,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D(2) (PGD(2))] induced formation of considerable peroxisome proliferator-activated receptor-gamma (PPARgamma) activity [Nature 391 (1998) 79]. Because PGD(2) itself is a poor PPARgamma ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D(2) for 24 h and studied the ability of the metabolites formed to activate PPARgamma. PGD(2) products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas-liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD(2) was converted to eight products, six of which were identified. Ligand-induced interaction of PPARgamma with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARgamma activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-delta(12,14)-PGJ(2)), a novel PPARgamma ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9alpha,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-delta(12,14)-PGD(2)) was identified. The biological significance of these results is currently under investigation.

  • 44.
    Toft, E
    et al.
    Wallenberg Laboratory, Stockholm University, Sweden.
    Becedas, L
    Wallenberg Laboratory, Stockholm University, Sweden.
    Söderström, Mats
    Wallenberg Laboratory, Stockholm University, Sweden.
    Lundqvist, A
    Wallenberg Laboratory, Stockholm University, Sweden.
    Depierre, J W
    Wallenberg Laboratory, Stockholm University, Sweden.
    Glutathione transferase isoenzyme patterns in the rat ovary1997In: Chemico-Biological Interactions, ISSN 0009-2797, E-ISSN 1872-7786, Vol. 108, no 1-2, p. 79-93Article in journal (Refereed)
    Abstract [en]

    This study was designed to investigate the expression of different isoenzymes of glutathione transferase (GST) in the rat ovary and to follow possible changes in the pattern of expression during maturation and the different stages of the oestrus cycle. The GST subunits present in the rat ovary (as analyzed by HPLC of affinity-purified GSTs) were A3, A4, M1, M2, M3 and P1. The most abundant subunit in the ovary was A3, but the mu enzymes demonstrated the largest increase (3.5-fold) when the mature ovary was compared to the immature organ. An overall decrease in GST isoenzyme content during dioestrus was observed, but there were no other recurrent changes during the oestrus cycle. Treatment of immature rats with pregnant mare's serum gonadotropin clearly demonstrated that the mu and alpha isoenzymes are up-regulated by gonadotropin stimulation, i.e. a 4-6 fold increase was seen. This treatment also elevated the P1 subunit 3-fold. At present, it is only possible to speculate concerning the mechanism(s) underlying these variations in ovarian GST expression in connection with hormonal changes. The functional significance of these variations is not yet known.

  • 45.
    Toft, E
    et al.
    Wallenberg Laboratory, Stockholm University, Sweden.
    Söderström, Mats
    Wallenberg Laboratory, Stockholm University, Sweden.
    Ahlberg, M B
    Wallenberg Laboratory, Stockholm University, Sweden.
    DePierre, J W
    Wallenberg Laboratory, Stockholm University, Sweden.
    A novel 34 kDa glutathione-binding protein in mature rat ovary1994In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 201, no 1, p. 149-154Article in journal (Refereed)
    Abstract [en]

    A novel protein that binds to a glutathione-Sepharose affinity column has been detected in mature, but not immature, rat ovary. This protein could be resolved from all identifiable components when the affinity-purified material, containing primarily glutathione transferases, was analyzed on reversed phase-HPLC. The unidentified protein migrated with an apparent molecular weight of 34 kDa on SDS-PAGE. After purification by affinity chromatography and subsequent preparative electrophoresis, the protein was subjected to N-terminal amino acid sequence analysis. The sequence obtained demonstrated a high degree of homology with an internal amino acid sequence in human carbonyl reductase (EC 1.1.1.184).

  • 46.
    Wahlström, Ola
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Orthopaedics in Linköping.
    Linder, Cecilia
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Kalén, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Orthopaedics in Linköping.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Per
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis2011In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 22, no 6, p. 452-460Article in journal (Refereed)
    Abstract [en]

    latelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p andlt;= 0.005, andgt;= 2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-beta in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

  • 47.
    Wahlström, Ola
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Orthopaedics in Linköping.
    Risto, Olof
    Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Orthopaedics in Linköping.
    Kalén, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Orthopaedics and Sports Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Orthopaedics in Linköping.
    Linder, Cecilia
    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.
    Ansell, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Per
    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.
    Acidic preparations of lysed platelets up-regulate proliferative and vascular genes, and the TGFBR pathway in osteoblast-like cells in BONE, vol 48, issue , pp S118-S1182011In: BONE, Elsevier Science B.V., Amsterdam. , 2011, Vol. 48, p. S118-S118Conference paper (Refereed)
    Abstract [en]

    n/a

  • 48.
    Wigren, Jane
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Surapureddi, Sailesh
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Olsson, Anders
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, MC - Medicincentrum, EMT-endo.
    Glass, C. K.
    University of California, San Diego, La Jolla, CA, USA.
    Hammarström, Sven
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Differential recruitment of the coactivator proteins CREB-binding protein and steroid receptor coactivator-1 to peroxisome proliferator-activated receptor gamma/9-cis-retinoic acid receptor heterodimers by ligands present in oxidized low-density lipoprotein2003In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 177, no 2, p. 207-214Article in journal (Refereed)
    Abstract [en]

    Peroxisome proliferator-activated receptor gamma (PPAR?) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPAR? activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxy-and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), as well as 5-hydroxy-, 12-hydroxy- and 15-hydroxyeicosatetraenoic acid (5-, 12- and 15-HETE respectively). PPAR? was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)- and 12(S)-HETE as well as 15-deoxy-?12,14 -prostaglandin J2 also activated PPAR? but were less potent. Interestingly, the effect of the lipoxygenase products 13(S -HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-?12,14-prostaglandin J2 was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPAR?/9-cis retinoic acid receptor heterodimer complexes.

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