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  • 1. Aleshkov, S B
    et al.
    Fa, M
    Karolin, J
    Strandberg, L
    Johansson, L B
    Wilczynska, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Biochemical and biophysical studies of reactive center cleaved plasminogen activator inhibitor type 1. The distance between P3 and P1' determined by donor-donor fluorescence energy transfer.1996In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 271, no 35, p. 21231-8Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

  • 2. Bergström, F
    et al.
    Hägglöf, P
    Karolin, J
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, L B
    The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.1999In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 22, p. 12477-81Article in journal (Refereed)
    Abstract [en]

    The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.

  • 3. Bergström, F
    et al.
    Hägglöf, P
    Karolin, J
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, L-B
    Application of donor-donor energy migration (DDEM) for examining protein structure and function1999In: Advances in Fluorescence Sensing Technology IV: Proceedings of SPIE / [ed] Lakowicz, J.R., Spoer, S.A., Thompson, R.B., SPIE , 1999, 3602, p. 250-255Chapter in book (Refereed)
  • 4. Bergström, Fredrik
    et al.
    Mikhalyov, Ilya
    Hägglöf, Peter
    Wortmann, Rüdiger
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B A
    Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology.2002In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 124, no 2, p. 196-204Article in journal (Refereed)
    Abstract [en]

    A ground-state dimer (denoted D(I)) exhibiting a strong absorption maximum at 477 nm (epsilon = 97 000 M(-1)cm(-1)) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited D(I) was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (D(I) and D(II)). Contrary to D(I) in PAI-1, the D(II) aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of D(I) is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (D(I)()) is 57 +/- 2 A. A simple model of exciton coupling suggests that in D(I) two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For D(II) the model suggests that the S(0) --> S(1) transition dipoles are colinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment (Delta(mu) approximately -0.05 D) and polarizability (-26 x 10(-40) C m(2) V(-1)) between the ground and the first excited states are small. Second, the S(0) <--> S(1) electronic transition dipole moments are perpendicular to Delta(mu).

  • 5.
    Bergström, Fredrik
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Mikhalyov, Ilya
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wortmann, Rüdiger
    Ny, Tor
    Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Dimers of Dipyrrometheneboron Difluoride (BODIPY) with Light Spectroscopic Applications in Chemistry and Biology2002In: Journal of the American Chemical Society, Vol. 124, no 2, p. 196-204Article in journal (Refereed)
    Abstract [en]

    A ground-state dimer (denoted DI) exhibiting a strong absorption maximum at 477 nm ( = 97 000 M-1cm-1) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited DI was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (DI and DII). Contrary to DI in PAI-1, the DII aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of DI is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (DI) is 57 ± 2 Å. A simple model of exciton coupling suggests that in DI two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For DII the model suggests that the S0 S1 transition dipoles are collinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment ( -0.05 D) and polarizability (-26 × 10-40 C m2 V-1) between the ground and the first excited states are small. Second, the S0 S1 electronic transition dipole moments are perpendicular to .

  • 6. Bicsak, T A
    et al.
    Cajander, S B
    Peng, X R
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    LaPolt, P S
    Lu, J K
    Kristensen, P
    Tsafriri, A
    Hsueh, A J
    Tissue-type plasminogen activator in rat oocytes: expression during the periovulatory period, after fertilization, and during follicular atresia.1989In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 124, no 1, p. 187-94Article in journal (Refereed)
    Abstract [en]

    The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.

  • 7. Casslén, B
    et al.
    Urano, S
    Lecander, I
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen activators in the human endometrium, cellular origin and hormonal regulation.1992In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235, E-ISSN 1473-5733, Vol. 3, no 2, p. 133-8Article in journal (Refereed)
    Abstract [en]

    Endometrial tissue explants in culture were found to release urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). In order to identify their cellular origin and possible hormonal regulation, enriched cultures of glandular epithelial cells and stromal cells were prepared from fresh endometrium, and the cultures treated with hormones. Both epithelial and stromal cell cultures were found to secrete u-PA and t-PA. Treatment of epithelial cell cultures with oestradiol, progesterone and DH-testosterone had no effect on the secretion of t-PA or u-PA. In stromal cell cultures, on the other hand, the secretion of u-PA was significantly reduced after treatment with progesterone, whereas oestradiol and DH-testosterone had no effect. This reduction of u-PA antigen in the tissue culture medium did not result from a reduction of the relative level of u-PA mRNA in the cells, suggesting that the synthesis of u-PA was not reduced. Alternatively, an increased clearance of u-PA by the cells from the medium may explain the reduction. This in vitro observation probably reflects the in vivo reduction of u-PA in endometrial secretion during the secretory phase.

  • 8. Casslén, B
    et al.
    Urano, S
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Progesterone regulation of plasminogen activator inhibitor 1 (PAI-1) antigen and mRNA levels in human endometrial stromal cells.1992In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 66, no 1, p. 75-87Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator activity decreases in the endometrium in the secretory phase of the menstrual cycle. This is partly due to decreased release of urokinase plasminogen activator in response to progesterone. Plasminogen activator inhibitor type 1 (PAI-1) is an efficient inhibitor of both tissue-type and urokinase-type plasminogen activators, and may therefore be instrumental for the control of plasminogen activation. In this study we examined the effects of steroid hormones on PAI-1 release and PAI-1 mRNA levels in primary cultures of human endometrial stromal cells. In these cells the secretion of PAI-1 was increased by progesterone in a dose and time dependent way, but was not affected by estradiol. The progesterone induction of PAI-1 secretion was preceded by a 7-8 fold increase of the steady state level of PAI-1 mRNA in the cells, suggesting that progesterone activates PAI-1 gene expression. Cultured endometrial glandular epithelial cells were found to release only insignificant amounts of PAI-1 with or without hormone treatment. The effect of progesterone on endometrial stromal cells was mimicked by DH-testosterone. However, while the response to progesterone was completely blocked by ZK112993, a potent antagonist of the progesterone receptor, the response to DH-testosterone was partially blocked by ZK112993, and partially by OH-flutamide, a potent antagonist of the androgen receptor. This suggests that a secretory response on PAI-1 expression is mediated via androgen receptors in endometrial tissue.

  • 9. Cheng, Fang
    et al.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Centre for Heart Lung Innovation, St. Paul ’ s Hospital, Vancouver, BC, Canada V6Z 1Y6; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada V6Z 1Y6.
    Mohanasundaram, Ponnuswamy
    Lindstrom, Michelle
    Ivaska, Johanna
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Eriksson, John E.
    Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-beta-Slug signaling2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 30, p. E4320-E4327Article in journal (Refereed)
    Abstract [en]

    Vimentin has been shown to be involved in wound healing, but its functional contribution to this process is poorly understood. Here we describe a previously unrecognized function of vimentin in coordinating fibroblast proliferation and keratinocyte differentiation during wound healing. Loss of vimentin led to a severe deficiency in fibroblast growth, which in turn inhibited the activation of two major initiators of epithelial-mesenchymal transition (EMT), TGF-beta 1 signaling and the Zinc finger transcriptional repressor protein Slug, in vimentin-deficient (VIM-/-) wounds. Correspondingly, VIM-/- wounds exhibited loss of EMT-like keratinocyte activation, limited keratinization, and slow reepithelialization. Furthermore, the fibroblast deficiency abolished collagen accumulation in the VIM-/- wounds. Vimentin reconstitution in VIM-/- fibroblasts restored both their proliferation and TGF-beta 1 production. Similarly, restoring paracrine TGF-beta-Slug-EMT signaling reactivated the transdifferentiation of keratinocytes, reviving their migratory properties, a critical feature for efficient healing. Our results demonstrate that vimentin orchestrates the healing by controlling fibroblast proliferation, TGF-beta 1-Slug signaling, collagen accumulation, and EMT processing, all of which in turn govern the required keratinocyte activation.

  • 10. Descheemaeker, K A
    et al.
    Wyns, S
    Nelles, L
    Auwerx, J
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Collen, D
    Interaction of AP-1-, AP-2-, and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myristate 13-acetate response.1992In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 267, no 21, p. 15086-91Article in journal (Refereed)
    Abstract [en]

    Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double-stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from Hela cells produced a gel shift pattern similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding, suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double-stranded -82 to -65 oligonucleotides with HeLa nuclear extracts revealed a gel shift pattern of three bands; Sp1 consensus oligomer competed with the binding to two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer spanning region -82 to -65 bound to proteins with molecular masses of 52 and 72 kDa. Consensus AP-2 oligonucleotides competed for binding of the labeled -82 to -65 oligonucleotide to the 52-kDa protein, but consensus Sp-1 oligonucleotides did not compete for binding to the 72-kDa compound. The 72-kDa component binding to the -82 to -65 region may represent a new protein involved in transcriptional regulation.

  • 11. Descheemaeker, K.A.
    et al.
    Nelles, L
    Strandberg, L
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Collen, D
    Transfection of human endothelial cells with human plasminogen activator inhibitor type-1 promoter-reporter gene fragmentes reveals phorbol ester induciton of gene expression1992In: Fibrinolysis, ISSN 0268-9499, Vol. 6, no 4, p. 256-262Article in journal (Refereed)
  • 12. Edlund, T
    et al.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rånby, M
    Hedén, L O
    Palm, G
    Holmgren, E
    Josephson, S
    Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.1983In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 80, no 2, p. 349-52Article in journal (Refereed)
    Abstract [en]

    We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.

  • 13. Erickson, L A
    et al.
    Schleef, R R
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Loskutoff, D J
    The fibrinolytic system of the vascular wall.1985In: Clinics in haematology, ISSN 0308-2261, Vol. 14, no 2, p. 513-30Article in journal (Refereed)
    Abstract [en]

    The vascular endothelium produces both PAs and a PAI. The activities of these components in the circulation must be regulated precisely to ensure that normal vascular homeostasis is not compromised. The blood contains a number of molecules that may function in this way by either promoting or inhibiting the synthesis, release and/or activity of the PAs and PAI. It is clear that the regulation of this system is considerably more complex than previously thought. For example, the initiation of fibrin dissolution is influenced by a number of additional factors including fibrin itself, pro-activators, PAI, platelet components (including the PAI), and possibly by APC generated at the endothelial cell surface. Despite the many recent advances discussed above, little is known about the temporal control of the events leading to plasminogen activation during thrombus formation and dissolution. Obviously, such information must be obtained before more effective treatments of abnormal vascular fibrinolytic activity can be developed. In this chapter, we have described a number of reagents and assays that should aid in the quantification of the PAs and the PAI in plasma. Eventual utilization of these assays in a clinical setting may be valuable for the diagnosis and subsequent treatment of abnormalities of the vascular fibrinolytic system.

  • 14.
    Eriksson, Per-Olof
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Li, Jinan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hellström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Spontaneous development of otitis media in plasminogen-deficient mice2006In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 296, no 7, p. 501-509Article in journal (Refereed)
    Abstract [en]

    Inflammatory conditions of the ear, otitis media, are one of the most common disease entities in children. In this study, the role of the plasminogen (plg)/plasmin system for the spontaneous development of chronic otitis media was investigated by the analysis of plg-deficient mice. Whereas essentially all of the wild-type control mice kept a healthy status of the middle ear, all the plg-deficient mice gradually developed chronic otitis media with various degrees of inflammatory changes during an 18-week observation period. Five bacterial strains were identified in materials obtained from the middle ear cavities of six plg-deficient mice. Morphological studies revealed the formation of an amorphous mass tissue and inflammatory changes in the middle ears of plg-deficient mice. Immunohistochemical studies further indicate a mass infiltration of neutrophils and macrophages as well as the presence of T and B cells in the middle ear mucosa of these mice. Extensive fibrin deposition and an abnormal keratin formation were also observed in the tympanic membrane, the middle ear cavity and external ear canal in these mice. These results suggest that plg plays an essential role in protecting against the spontaneous development of chronic otitis media. Our findings also suggest the possibility of using plg for clinical therapy of certain types of otitis media.

  • 15. Fa, M.
    et al.
    Bergström, F.
    Hägglöf, P.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, M.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, L. B.-A.
    Ny, T.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites2000In: Structure, with Folding & Design, Vol. 8, no 4, p. 397-405Article in journal (Refereed)
  • 16. Fa, M
    et al.
    Bergström, F
    Karolin, J
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 12, p. 3729-34Article in journal (Refereed)
    Abstract [en]

    The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

  • 17. Fa, M
    et al.
    Karolin, J
    Aleshkov, S
    Strandberg, L
    Johansson, L B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.1995In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 34, no 42, p. 13833-40Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

  • 18.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites2000In: Structure, Vol. 8, no 4, p. 397-405Article in journal (Refereed)
    Abstract [en]

    Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.

    Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.

    Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

  • 19.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy: Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms2000In: European Journal of Biochemistry, Vol. 267, no 12, p. 3729-34Article in journal (Refereed)
    Abstract [en]

    The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY® probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57 ± 4 Å and 63 ± 3 Å, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into β-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

  • 20.
    Fallah, Mahsa
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Brodén, Jessica
    Bäckman, Assar
    Lundskog, Bertil
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Johansson, Mikael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Blomqvist, Michael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Liu, Kui
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen activation is required for the development of radiation-induced dermatitis2018In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 9, no 11, article id 1051Article in journal (Refereed)
    Abstract [en]

    Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild type (plg+/+) mice, the plasminogen level increased in the radiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg−/−) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/ mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

     

     

  • 21.
    Fallah, Mahsa
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Viklund, Emil
    Shen, Yue
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bäckman, Assar
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundskog, Bertil
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Johansson, Mikael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Blomqvist, Michael
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Liu, Kui
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Plasminogen enhances the healing of radiation-induced wounds via decreased expression of pro-inflammatory and pro-fibrotic factorsManuscript (preprint) (Other academic)
  • 22. Feng, P
    et al.
    Ohlsson, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The structure of the TATA-less rat tissue-type plasminogen activator gene. Species-specific sequence divergences in the promoter predict differences in regulation of gene expression.1990In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 265, no 4, p. 2022-7Article in journal (Refereed)
    Abstract [en]

    The genomic region carrying the rat tissue-type plasminogen activator (tPA) gene including its 5'-flanking sequence has been isolated and characterized by restriction enzyme analysis, Southern blotting, and DNA sequencing of all coding parts and the promoter region. The gene is approximately 25 kilobase pairs in size and comprises 14 exons separated by 13 introns. All the exon/intron boundaries agree with the GT-AG rule. The organization of the rat tPA gene is very similar to its human counterpart, and the location of the introns in the protein structure is identical to the human tPA gene. To characterize the promoter region, the transcription initiation site was identified by S1 nuclease protection experiments. A DNA fragment carrying 621 nucleotides of the 5'-flanking sequence was found to confer basal promoter activity and hormone responsiveness to a reporter gene construct in primary cultures of rat granulosa cells. Analysis of the rat tPA promoter sequence and a comparison with the human and mouse counterparts reveal several species-specific differences: the rat and mouse tPA promoters lack typical TATA and CAAT sequences found in the human tPA gene. Furthermore, the rat tPA promoter contains a consensus cAMP-responsive element shown to be required for cAMP responsiveness in eucaryotic genes. At the same position as the cAMP-responsive element in the rat gene, the mouse and human tPA genes have a 12-O-tetradecanoylphorbol-13-acetate-responsive element known to mediate activation by phorbol esters. The differences in the promoter sequences of the rat, mouse, and human tPA genes may have implications for the regulation of the tPA gene in different species.

  • 23. Galway, A B
    et al.
    Oikawa, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hsueh, A J
    Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C.1989In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 125, no 1, p. 126-35Article in journal (Refereed)
    Abstract [en]

    Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.

  • 24. Glise, Lars
    et al.
    Larsson, Pia
    Jern, Sverker
    Borén, Jan
    Levin, Malin
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Fogelstrand, Per
    Bergh, Niklas
    Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo2019In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 119, no 2, p. 223-233Article in journal (Refereed)
    Abstract [en]

    Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.

  • 25.
    Grimsholm, Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Yongzhi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Expression patterns of neurotrophins and neurotrophin receptors in articular chondrocytes and inflammatory infiltrates in knee joint arthritis.2008In: Cells, tissues, organs, ISSN 1422-6421, Vol. 188, no 3, p. 299-309Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: It is likely that neurotrophins (NTs) are of great importance for the articular cartilage and the inflammation process in arthritis. METHODS: The immunohistochemical expression of the NTs nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and the associated receptors p75, TrkA and TrkB was examined in the knee joint of arthritic and healthy mice. RESULTS: Immunoreactions for NGF and BDNF were detected in cells and nerve fiber varicosities in the inflammatory infiltrates of the synovial tissue of arthritic joints but not in synovial tissue of controls. p75-immunoreactive nerve fiber-like strands were detected in inflammatory infiltrates. Immunostaining for NGF, BDNF, p75, TrkA and TrkB was noted in articular chondrocytes. There was a statistically significant decrease in reactions for NGF (p < 0.001), TrkA (p = 0.001) and p75 (p < 0.001) in articular chondrocytes in joints exhibiting severe arthritis. CONCLUSION: The findings show that an NT system develops in inflammatory infiltrates of the synovial tissue. Furthermore, most interestingly, autocrine/paracrine effects appear to exist concerning NTs for the articular chondrocytes. The downregulated expression of NGF and NT receptors in articular chondrocytes in arthritis is a new aspect concerning the involvement of NTs in cartilage.

  • 26.
    Grimsholm, Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Yongzhi
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Rantapää-Dahlqvist, Solbritt
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Are neuropeptides important in arthritis? Studies on the importance of bombesin/GRP and substance P in a murine arthritis model.2007In: Annals of the New York Academy of Sciences, ISSN 0077-8923, E-ISSN 1749-6632, Vol. 1110, p. 525-538Article in journal (Refereed)
    Abstract [en]

    Interference with the effects of neuropeptides may be of potential therapeutic value for the treatment of rheumatoid arthritis (RA). Two neuropeptides that can be discussed in this context are bombesin/gastrin-releasing peptide (BN/GRP) and substance P (SP). In order to obtain new information on the possible importance of these two peptides, the patterns of immunohistochemical expression of BN/GRP and SP and their related receptors in the mouse knee joint from healthy and arthritic mice were examined. Positive staining for GRP receptor and the SP preferred receptor (the neurokinin-1 receptor [NK-1 R]) was observed in articular chondrocytes. On the whole, there was a decrease in immunoreactions for both the GRP- and the NK-1 receptors in the articular chondrocytes in joints exhibiting severe arthritis. Staining for BN/GRP and GRP receptor was seen in the inflammatory infiltrates of the arthritic joints. New evidence for the occurrence of marked effects of BN/GRP concerning both the articular chondrocytes and the inflammatory process is obtained in this study. With these findings and previous observations of neuropeptide expression patterns and functions we discuss the possibility that interventions with the effects of BN/GRP, SP, and other neuropeptides might be worthwhile in RA.

  • 27.
    Guo, Yong-Zhi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Li, Jinan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hagstrom, Elin
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Beneficial and detrimental effects of plasmin(ogen) during infection and sepsis in mice2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 9, p. e24774-Article in journal (Refereed)
    Abstract [en]

    Plasmin has been proposed to be an important mediator during inflammation/infection. In this study, by using mice lacking genes for plasminogen, tissue-type plasminogen activator (tPA), and urokinase-type PA (uPA), we have investigated the functional roles of active plasmin in infection and sepsis. Two models were used: an infection model by intravenous injection of 1x10(7) CFU of S. aureus, and a sepsis model by intravenous injection of 1.6x10(8) CFU of S. aureus. We found that in the infection model, wild-type (WT) mice showed significantly higher survival rates than plasminogen-deficient (plg(-/-)) mice. However, in the sepsis model, plg(-/-) or tPA(-/-)/uPA(-/-) mice showed the highest survival rate whereas WT and tPA(+/-)/uPA(+/-) mice showed the lowest survival rate, and plg(+/-), tPA(-/-), and uPA(-/-) mice had an intermediate survival rate. These results indicate that the levels of active plasmin are critical in determining the survival rate in the sepsis, partly through high levels of inflammatory cytokines and enhanced STAT3 activation. We conclude that plasmin is beneficial in infection but promotes the production of inflammatory cytokines in sepsis that may cause tissue destruction, diminished neutrophil function, and an impaired capacity to kill bacteria which eventually causes death of these mice.

  • 28.
    Guo, Yongzhi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Li, Jinan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hagström, Elin
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Beneficial and detrimental effects of plasmin(ogen) during infection and sepsisArticle in journal (Refereed)
  • 29.
    Guo, Yongzhi
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Li, Jinan
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hagström, Elin
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Protective effects of plasmin(ogen) in a mouse model of Staphylococcus aureus-induced arthritis2008In: Arthritis and rheumatism, ISSN 0004-3591, Vol. 58, no 3, p. 764-772Article in journal (Refereed)
    Abstract [en]

    Objective: To assess the functional roles of plasmin in a murine model of Staphylococcus aureus-induced bacterial arthritis. Methods: Bacterial arthritis was induced in plasminogen-deficient (Plg-/-) and wild-type (Plg+/+) littermates by local injection of 1 × 106 colony-forming units of S aureus into the knee joints. Human plasminogen was administered to Plg-/- mice on days 0-7 or days 7-14. Antibiotic treatment was administered to Plg-/- mice on days 7-14. Bacteria counts and histologic, immunohistochemical, and Western blot analyses were performed. Results: In Plg+/+ mice, S aureus counts had declined within 2 days, and by day 28 the bacteria had been completely eliminated. However, S aureus was still detectable in all injected joints from Plg-/- mice, and bacteria counts were 27 times higher than the amount injected on day 0. The extent of macrophage and neutrophil recruitment to the infected joints was comparable for Plg+/+ and Plg-/- mice on days 1, 7, and 14. The activation of these inflammatory cells appeared to be impaired in Plg-/- mice, however. Treatment of Plg-/- mice with antibiotic (cloxacillin) resulted in successful killing of the bacteria, but the necrotic tissue remained in the infected joints. When human plasminogen was given intravenously to Plg-/- mice daily for 7 days, bacterial clearance was greatly improved as compared with their untreated counterparts, and the amount of necrotic tissue in the joint cavity was dramatically reduced. The expression of interleukin 6 (IL-6) and IL-10 was higher in Plg+/+ mice than in Plg-/- mice during bacterial arthritis. Conclusion: Our findings indicate that plasmin plays a pluripotent role in protecting against S aureus-induced arthritis by activating inflammatory cells, killing bacteria, removing necrotic tissue, and enhancing cytokine expression.

  • 30. Gustafsson, A
    et al.
    Sundström, S
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundgren, E
    Rapid induction of seven proteins in human lymphocytes by interferon; correlation to natural killer cell activity.1982In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 129, no 5, p. 1952-9Article in journal (Refereed)
    Abstract [en]

    The early effects of interferon (IFN) on the synthesis of protein in human nylon wool-nonadherent lymphocytes have been stimulated by use of two-dimensional electrophoresis. IFN-alpha or -beta as well as Escherichia coli-produced IFN-alpha 2 induced the rapid formation of seven proteins (Mr 80, 75, 62, 53, 38, 36, and 33 kD). At least five proteins were expressed within 2 hr of incubation with IFN. The synthesis of the seven proteins seemed to require rapid transcription of new RNA, because actinomycin D markedly inhibited their formation only when added less than 30 min after IFN. A good correlation was found between the ability of actinomycin D to inhibit both the formation of new proteins and the augmentation of natural killer (NK) cell activity. Screening of a panel of 10 hematopoietic and two anchorage-dependent cell lines revealed that p62 and p38 were induced in most cell lines, whereas p80 and p33 were preferentially induced in lymphoid cell lines. Three proteins could not be induced by IFN in any of the 12 cell lines, and thus could represent molecules mediating differentiated functions, possibly involved in NK cell function.

  • 31.
    Hansson, Annika
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Hellström, Sten
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Eriksson, Per-Olof
    Li, Jinan
    Berggren, Diana
    Plasminogen deficiency does not alter the healing of tympanic membrane perforations in vitroManuscript (Other academic)
  • 32.
    Hansson, Annika
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Li, Jinan
    Eriksson, Per-Olof
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Berggren, Diana
    Hellström, Sten
    The healing of tympanic membrane perforations is unaffected by urokinase-type plasminogen activator deficiencyManuscript (Other academic)
  • 33.
    Hellström, Sten
    et al.
    Department of CLINTEC/Otorhinolaryngology, Karolinska Medical University.
    Shen, Yue
    University of British Columbia.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    A reply to the commentary on "Animal models of chronic tympanic membrane perforation: in response to plasminogen initiates and potentiates the healing of acute and chronic tympanic membrane perforations in mice" by Wang AY, Shen Y, Wang JT, Eikelboom RH and Dilley RJ; Clin Translat Med, 2014; 32015In: Clinical and translational medicine, ISSN 2001-1326, Vol. 4, no 8Article in journal (Refereed)
  • 34. Hepburn, Lucy
    et al.
    Prajsnar, Tomasz K
    Klapholz, Catherine
    Moreno, Pablo
    Loynes, Catherine A
    Ogryzko, Nikolay V
    Brown, Karen
    Schiebler, Mark
    Hegyi, Krisztina
    Antrobus, Robin
    Hammond, Katherine L
    Connolly, John
    Ochoa, Bernardo
    Bryant, Clare
    Otto, Michael
    Surewaard, Bas
    Seneviratne, Suranjith L
    Grogono, Dorothy M
    Cachat, Julien
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kaser, Arthur
    Török, M Estée
    Peacock, Sharon J
    Holden, Matthew
    Blundell, Tom
    Wang, Lihui
    Ligoxygakis, Petros
    Minichiello, Liliana
    Woods, C Geoff
    Foster, Simon J
    Renshaw, Stephen A
    Floto, R Andres
    A Spaetzle-like role for nerve growth factor beta in vertebrate immunity to Staphylococcus aureus2014In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 346, no 6209, p. 641-646Article in journal (Refereed)
    Abstract [en]

    Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRP4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity.

  • 35. Holmberg, M
    et al.
    Leonardsson, G
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The species-specific differences in the cAMP regulation of the tissue-type plasminogen activator gene between rat, mouse and human is caused by a one-nucleotide substitution in the cAMP-responsive element of the promoters.1995In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 231, no 2, p. 466-74Article in journal (Refereed)
    Abstract [en]

    In rat ovarian cells tissue-type plasminogen activator (tPA) is induced by gonadotropins, by a cAMP-dependent pathway and the induction correlates with the time of follicle rupture in vivo. However, in mice, gonadotropins induce the related but distinct protease urokinase-type plasminogen activator (uPA). Comparison of rat, mouse and human tPA genes reveal that there is a species-specific difference in the promoter that could explain the difference in regulation of the tPA gene between these species. At the position where the rat promoter contains a consensus cAMP-responsive element (CRE), the mouse and human counterparts contains a CRE variant with a one-nucleotide substitution. Transient transfection experiments of rat glial and granulosa cells demonstrated that reporter constructs driven by rat but not mouse or human tPA promoters were efficiently induced by the cAMP-inducing agents forskolin or follicle-stimulating hormone. Following the conversion of the mouse and human CRE-like sequences to rat consensus CRE these promoters became cAMP responsive. In contrast the rat promoter, following conversion of the consensus CRE to the corresponding mouse and human CRE-like sequence, lost the ability to efficiently respond to cAMP. Deoxyribonuclease I footprinting analysis and electrophoretic mobility shift assays were used to examine interactions of nuclear factors with the consensus and variant CRE. Compared to rat CRE, the mouse and human CRE-like sequences had a drastically reduced binding affinity for a nuclear factor identified as the cAMP-responsive element binding protein. Thus the inability of the mouse and human tPA promoters to respond efficiently to forskolin and follicle-stimulation hormone seem to be due to the inability of these CRE-like sequences to efficiently bind transcription factor CRE binding protein.

  • 36.
    Hosokawa, K
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Ottander, Ulrika
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Wahlberg, P
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Cajander, S
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Olofsson, Jan I
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Dominant expression and distribution of oestrogen receptor beta over oestrogen receptor alpha in the human corpus luteum2001In: Molecular human reproduction, ISSN 1360-9947, E-ISSN 1460-2407, Vol. 7, no 2, p. 137-145Article in journal (Refereed)
    Abstract [en]

    To investigate the potential importance of oestrogen as a local regulator of human corpus luteum function, the mRNA expression pattern and cellular localization of oestrogen receptors (ERs), ER-alpha and ER-beta, were studied in corpora lutea grouped according to age, where days 2-5 post-LH rise were designated as the early luteal phase, days 6-10 as mid-luteal and days 11-14 as the late luteal phase respectively. Northern blot analysis using an ER-beta probe in samples from whole ovarian tissue and isolated corpora lutea, revealed a major band at 7.5 kb and several minor bands between 4-10 kb, while no signals for ER-alpha mRNA were obtained. However, using a semi-quantitative reverse transcription-polymerase chain reaction followed by Southern blotting, ER-beta mRNA levels were found to be 63% lower (P: < 0.05, n = 39) in the mid-luteal phase compared with the early luteal phase, while ER-alpha mRNA expression showed no statistical differences between the different age groups. Using in-situ hybridization, ER-beta mRNA expression was localized to the steroidogenic luteal cells as well as perivascular cells and fibroblasts in the corpus luteum. Immunohistochemistry confirmed the localization of ER-beta protein, but no clear staining of luteal cells was found using antibodies against ER-alpha. Collectively, the findings of low to moderate expression of ER-beta mRNA and protein in the steroidogenic cells, and also in vascular endothelial cells of the corpus luteum, as opposed to diminutive amounts of ER-alpha mRNA, suggest that oestrogen activity is primarily transduced via ER-beta in the human corpus luteum.

  • 37. Hsueh, A J
    et al.
    Liu, Y X
    Cajander, S B
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Molecular mechanisms in the hormonal regulation of plasminogen activator activity in ovarian granulosa cells and cumulus-oocyte complexes.1988In: Progress in clinical and biological research, ISSN 0361-7742, Vol. 267, p. 227-57Article in journal (Refereed)
  • 38. Hsueh, A J
    et al.
    Liu, Y X
    Cajander, S
    Peng, X R
    Dahl, K
    Kristensen, P
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization.1988In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 122, no 4, p. 1486-95Article in journal (Refereed)
    Abstract [en]

    GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.

  • 39. Hsueh, A.J.W.
    et al.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    GnRH as an ovulation-inducing agent: Regulation of plasminogen activator activity1985In: Proceedings of the 11th World Congress of Gynecology and Obstetrics, Springer-Verlag New York, 1985Conference paper (Refereed)
  • 40. Hu, Z Y
    et al.
    Liu, Y X
    Liu, K
    Byrne, S
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Feng, Q
    Ockleford, C D
    Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta.1999In: Journal of Anatomy, ISSN 0021-8782, E-ISSN 1469-7580, Vol. 194 ( Pt 2), p. 183-95Article in journal (Refereed)
    Abstract [en]

    The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type-1 (PAI-1) and type-2 (PAI-2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI-1 and PAI-2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI-1 and PAI-2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohr's and Nitabuch's striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI-2 was noted in villous trophoblast whereas tPA and PAI-1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI-1 and PAI-2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI-1 and PAI-2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI-1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI-2 appears mainly to play a role in degradation of trophoblast cell-associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

  • 41. Hägglund, A C
    et al.
    Basset, P
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Stromelysin-3 is induced in mouse ovarian follicles undergoing hormonally controlled apoptosis, but this metalloproteinase is not required for follicular atresia.2001In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 64, no 2, p. 457-63Article in journal (Refereed)
    Abstract [en]

    Apoptotic processes are often associated with an intense proteolytic remodeling of the extracellular matrix (ECM). Proteolytic degradation of the ECM can also be a signal that induces apoptosis. Here, we have investigated the expression pattern and functional role of the matrix metalloproteinase stromelysin-3 in follicular atresia. Twenty-four hours after the treatment of immature female mice with a low dose of eCG, both apoptosis and the stromelysin-3 mRNA expression were suppressed approximately threefold. However, the initial suppression of apoptosis and stromelysin-3 expression was followed by a time-dependent increase, and 96 h after eCG treatment, the levels were similar to those of untreated control mice. In 15- to 16-day-old juvenile mice, the ovary consisted of relatively undeveloped follicles, and almost no apoptosis and only low stromelysin-3 mRNA expression were observed. However, at the age of 21 days, when several antral follicles were present, a fivefold induction in both apoptosis and stromelysin-3 mRNA expression was detected. For both models, in situ analysis revealed that the expression of stromelysin-3 mRNA was localized to the granulosa cells of atretic follicles. To address the functional role of stromelysin-3 in follicular atresia, stromelysin-3-deficient mice were studied. However, no difference in the pattern of apoptotic DNA fragmentation and no apparent morphological differences were observed when ovaries from wild-type and stromelysin-3-deficient mice were compared. Taken together, our data indicate that stromelysin-3 is induced during follicular atresia, but that this protease is not obligatory for initiation or completion of the atretic process.

  • 42. Hägglund, A C
    et al.
    Ny, A
    Leonardsson, G
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation and localization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse ovary during gonadotropin-induced ovulation.1999In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 140, no 9, p. 4351-8Article in journal (Refereed)
    Abstract [en]

    At the time of ovulation, proteolytic degradation of the follicular wall is required to release the mature oocyte. Extracellular proteases, such as serine proteases and matrix metalloproteinases (MMPs), are thought to play important roles in this process. In this study we have examined the regulation of 11 MMPs and 3 tissue inhibitors of metalloproteinases (TIMPs) during gonadotropin-induced ovulation in the mouse. Northern blot hybridization showed that messenger RNA for several MMPs and TIMPs, including gelatinase A, MT1-MMP, stromelysin-3, MMP-19, TIMP-1, TIMP-2, and TIMP-3, were present at detectable levels in the mouse ovary. In addition, ovarian extracts contained gelatinolytic activities corresponding to the inactive proforms of gelatinase A and gelatinase B. Most of the MMPs and TIMPs were expressed at a constitutive level throughout the periovulatory period. However, MMP-19 and TIMP-1 revealed a different expression pattern; they were both induced 5-10 times by hCG and reached their maximum levels at 12 h after hCG treatment, corresponding to the time of ovulation. At this time point, MMP-19 and TIMP-1 messenger RNA were localized to the granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. This temporal and spatial regulation pattern suggests that MMP-19 might be involved in the tissue degradation that occurs during follicular rupture and that TIMP-1 could have a role in terminating MMP activity after ovulation.

  • 43. Hägglund, A C
    et al.
    Ny, A
    Liu, K
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation.1996In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 137, no 12, p. 5671-7Article in journal (Refereed)
    Abstract [en]

    Several lines of indirect evidence indicate that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation. Consistent with this, the ovulation efficiency of mice lacking the two known physiological plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), is reduced by 26%. Surprisingly, mice with a single deficiency of either tPA or uPA gene function were normal in their capacity to ovulate. In this study we used in situ hybridization and casein in situ zymography to localize the expression of messenger RNAs (mRNAs) encoding PAs and PA inhibitors and to examine the net PA activity in the mouse ovary at the time of ovulation. Although uPA mRNA expressed by granulosa cells is the most abundant and dramatically up-regulated PA before ovulation, a previously unnoticed coordinated induction oftPA mRNA was found in thecal-interstitial tissue. The existence of redundant mechanisms for plasmin production in the ovary may be the cause of the normal ovulation efficiency in single deficient mice lacking tPA or uPA. The expression of mRNAs for PA inhibitors, types 1 and 2, was low in the ovary, with minor inductions at restricted time points. In contrast, expression of protease nexin-1 (PN-1) by granulosa cells was high during the entire periovulatory period. Among subpopulations of granulosa cells, the expression of PN-1 and uPA was heterogeneous and complementary. Cumulus cells expressed high levels of PN-1 mRNA and low levels of uPA mRNA, thereby providing an inhibitory activity that may protect the mucified matrix of the cumulus oocyte complex from proteolytic degradation.

  • 44.
    Hägglöf, Peter
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 3, p. 823-832Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

  • 45.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Håkansson, Pär
    Umeå University, Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Extended Förster theory for determining intraprotein distances: 2. an accurate analysis of fluorescence depolarisation experiments2007In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 9, p. 3914-3922Article in journal (Refereed)
    Abstract [en]

    The extended Förster theory (EFT) is for the first time applied to the quantitative determination of the intramolecular distances in proteins. It is shown how the EFT (J. Chem. Phys., 1996, 105, 10896) can be adapted to the analyses of fluorescence depolarisation experiments based on the time-correlated single photon counting technique (TCSPC). The protein system studied was the latent form of plasminogen activator inhibitor type I (PAI-1), which was mutated and labelled by the thiol reactive BODIPY® derivative {N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide}. The energy migration occurs within pairs of photophysically identical donor groups that undergo reorientational motions on the timescales of energy migration and fluorescence relaxation. Unlike all models currently used for analysing fluorescence TCSPC data, the EFT explicitly accounts for the time-dependent reorientations that influence the rate of electronic energy transfer/migration in a complex manner. The complexity is related to the 2 problem, which has been discussed for years. The EFT brings the analyses of DDEM data to the same level of molecular description as in ESR and NMR spectroscopy, i.e. it yields microscopic information about the reorientation correlation times, the order parameters, as well as inter-chromophoric distances.

  • 46.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lobov, Sergei
    Umeå University, Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    An environmental-sensitive BODIPY®-derivative with bioapplication: spectral and photophysical properties2003In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 13, no 5, p. 379-84Article in journal (Refereed)
    Abstract [en]

    A previously synthesised derivative of BODIPY aimed for sulfhydryl specific labelling of cysteine residues in proteins was studied. The spectral and photophysical properties of this derivative, N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide (NBDY) were characterised, and found to be considerably different from those of commonly used derivatives of BODIPY, e.g. N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide. The absorption and fluorescence spectra, as well as fluorescence lifetimes and quantum yields of NBDY are quite sensitive to solvent properties. The fluorescence is effectively quenched by I– when NBDY is free in water or attached to Cys in different mutants of plasminogen activator inhibitor type 2 (PAI-2). A ground-state dimer forms when two NBDY groups are closely spaced in plasminogen activator inhibitor type 1 (PAI-1).

  • 47.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lobov, Sergei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wang, Shouye
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements2004In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 6, no 11, p. 3001-3008Article in journal (Refereed)
    Abstract [en]

    We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor-donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-Angstrom. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1057-1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely: N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4.4-difluoro-5.7-ditriethyl-4-bona-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor-donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of polyfluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.

  • 48. Jia, X C
    et al.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hsueh, A J
    Synergistic effect of glucocorticoids and androgens on the hormonal induction of tissue plasminogen activator activity and messenger ribonucleic acid levels in granulosa cells.1990In: Molecular and Cellular Endocrinology, ISSN 0303-7207, E-ISSN 1872-8057, Vol. 68, no 2-3, p. 143-51Article in journal (Refereed)
    Abstract [en]

    Tissue-type plasminogen activator (tPA) is secreted by rat granulosa cells in response to treatment with activators of protein kinase A (follitropin, FSH), protein kinase C (gonadotropin-releasing hormone, GnRH) and tyrosine kinase (epidermal growth factor, EGF). Because steroid hormones have been shown to enhance the gonadotropin stimulation of ovarian differentiation, we investigated the effects of steroid hormones, alone or together with various kinase activators, on tPA activities and mRNA levels in cultured rat granulosa cells. Treatment of cells with dexamethasone (DEX; a glucocorticoid agonist) or R1881 (an androgen agonist) caused an increase in tPA secretion and mRNA levels. In addition, the stimulation of tPA activity and mRNA levels by FSH (50 ng/ml) was synergistically enhanced by cotreatment with DEX or R1881 in a time-dependent manner with 2.8- and 1.6-fold increase at 9 h after incubation as compared to cells treated with FSH alone. In contrast, treatment with diethylstilbestrol had no effect on tPA levels. Furthermore, tPA activity and mRNA levels induced by GnRH and EGF were also increased by cotreatment with DEX or R1881 as compared with cells treated with GnRH or EGF alone. Likewise, the stimulation of tPA mRNA levels by dibutyryl cAMP, a protein kinase A activator, and phorbol myristate acetate (PMA), a protein kinase C activator, was enhanced by cotreatment with DEX or R1881. These results demonstrate that glucocorticoid and androgen enhance tPA secretion and mRNA levels stimulated by FSH, GnRH and EGF in granulosa cells. The rat granulosa cells provide a useful model for studying the mechanism of regulation of tPA gene expression by steroid hormones.

  • 49. Jia, X C
    et al.
    Oikawa, M
    Bo, M
    Tanaka, T
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Boime, I
    Hsueh, A J
    Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species.1991In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 5, no 6, p. 759-68Article in journal (Refereed)
    Abstract [en]

    Studies on human LH receptors are difficult due to the limited availability of clinical samples. Recent cloning of rat and porcine LH receptor cDNAs indicated that these binding sites are single polypeptides of the G-protein-coupled receptor family with seven transmembrane domains. Based on the conserved sequences of rat and porcine receptors, we performed reverse transcription polymerase chain reaction, using human ovarian mRNA as template and obtained partial human LH receptor cDNA clones. Further screening of a human ovary cDNA library and subsequent ligation of individual cDNA clones generated a human LH receptor cDNA containing the entire amino acid-coding region. Sequence analysis indicated that the human receptor cDNA displays 89% and 82% homology at the nucleotide level with its porcine and rat counterparts, respectively. A region spanning the second extracellular and third transmembrane domains is highly conserved among the human LH, FSH, and TSH receptors. The ovarian LH receptor clone is, however, significantly different from an incompletely spliced LH receptor cDNA recently obtained from a human thyroid library. Unlike the thyroid clone, the ovarian LH receptor cDNA could be expressed in the human fetal kidney cell line (293), and radioligand receptor assay identified high affinity (Kd, 1.2 x 10(-10) M) LH/hCG-binding sites on the plasma membrane. Binding specificity of the human LH receptor was studied using recombinant human CG, LH, and FSH secreted by CHO cells transfected with the respective genes. Human CG and LH displaced [125I]hCG binding with an ED50 of 4.3 and 4.8 ng/ml, respectively. In contrast, recombinant FSH was not effective. Treatment of transfected cells with recombinant gonadotropins also induced dose-dependent increases in extracellular cAMP production (hCG = LH much greater than FSH; ED50 25, 10, and greater than 3000 ng/ml). Although equine, rat, and ovine LH as well as equine CG competed effectively for rat testicular LH receptor binding, these hormones were unable to displace [125I]hCG binding to the human receptor, suggesting evolutionary changes in receptor binding specificity and the importance of using human receptors for clinical studies. Thus, the cloning and expression of the human LH receptor cDNA allowed analysis of interactions between human LH receptor and gonadotropins from diverse species. The present work should provide the basis for future design of therapeutic agents capable of interacting with the human receptor and for understanding the structural basis for LH receptor binding to different gonadotropins.

  • 50. Jia, X C
    et al.
    Perlas, E
    Su, J G
    Moran, F
    Lasley, B L
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hsueh, A J
    Luminescence luteinizing hormone/choriogonadotropin (LH/CG) bioassay: measurement of serum bioactive LH/CG during early pregnancy in human and macaque.1993In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 49, no 6, p. 1310-6Article in journal (Refereed)
    Abstract [en]

    Because of the microheterogeneities of gonadotropins, measurement of immunoreactivity of these glycoproteins does not necessarily reflect changes in their bioactivity. In addition, LH bioactivities in human samples analyzed by a rodent LH bioassay have been discordant with findings based on human granulosa-luteal cells. We have isolated a human LH/choriogonadotropin (CG) receptor cDNA and expressed the recombinant protein. Using 293 cells permanently transfected with the human LH receptor cDNA and a luciferase reporter gene driven by a cAMP-dependent promoter, we have developed a luminescence LH/CG bioassay. After cells were treated with human LH or CG for 20 h, luciferase activity was measured through use of a luminometer. Luciferase activity in the cells was increased in a dose-dependent manner. In contrast, treatment with FSH, thyroid-stimulating hormone, prolactin, growth hormone, adrenocorticotropin, insulin, prostaglandins, and several neurotransmitters had no effect. Because treatment with basic fibroblast growth factor (bFGF) caused significant increases in basal luciferase activity, a fixed amount of bFGF was included in all reactions. Incubation with 0.1 to 30 microliters serum from women during different physiological states stimulated the luciferase activity in parallel with the hCG standard curve. In 4 conception cycles, bioactive LH/hCG levels began to increase 2 wk after the midcycle LH surge, followed by a logarithmic increase from 22 days on. Due to the lack of a homologous RIA for measuring CG levels in monkeys, we analyzed serum bioactive monkey CG (mCG) in macaque during early pregnancy. Bioactive mCG was detected about 12 days after the midcycle LH surge and fertile mating and persisted until Days 21-23, followed by a decline.(ABSTRACT TRUNCATED AT 250 WORDS)

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