| 1. |
- Aleshkov, S B, et al.
(författare)
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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.
- 1996
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 271:35, s. 21231-8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
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| 3. |
- Bergström, Fredrik, et al.
(författare)
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Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology.
- 2002
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Ingår i: Journal of the American Chemical Society. - 0002-7863 .- 1520-5126. ; 124:2, s. 196-204
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 4. |
- Bergström, Fredrik, et al.
(författare)
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Dimers of Dipyrrometheneboron Difluoride (BODIPY) with Light Spectroscopic Applications in Chemistry and Biology
- 2002
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:2, s. 196-204
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Tidskriftsartikel (refereegranskat)abstract
- 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 .
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| 5. |
- Bergström, F, et al.
(författare)
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The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.
- 1999
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 96:22, s. 12477-81
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Bicsak, T A, et al.
(författare)
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Tissue-type plasminogen activator in rat oocytes : expression during the periovulatory period, after fertilization, and during follicular atresia.
- 1989
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 124:1, s. 187-94
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Borg, H, et al.
(författare)
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Evidence for IFN-beta heterogeneity in a substrain of Namalwa cells.
- 1985
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Ingår i: Applied Biochemistry and Biotechnology. - 0273-2289 .- 1559-0291. ; 11:2, s. 111-22
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Tidskriftsartikel (refereegranskat)abstract
- A substrain of Namalwa cells, denoted substrain B, was grown in fermentors up to the 100-L scale, and was induced with Sendai virus to produce interferon (IFN). The titer of the crude IFN varied extensively between different batches; part of the variation was caused by a differential expression of IFN-alpha and IFN-beta. More than 80% of the IFN activity was IFN-beta by several criteria. A two-step purification procedure was developed and the resulting preparation had a specific activity of approximately 10(6) U/mg protein. The IFN-beta type was found to be heterogeneous, and could be separated into several components, which probably represented post-translational modifications of one molecule.
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| 8. |
- Casslén, B, et al.
(författare)
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Plasminogen activators in the human endometrium, cellular origin and hormonal regulation.
- 1992
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Ingår i: Blood Coagulation and Fibrinolysis. - 0957-5235 .- 1473-5733. ; 3:2, s. 133-8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Casslén, B, et al.
(författare)
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Progesterone regulation of plasminogen activator inhibitor 1 (PAI-1) antigen and mRNA levels in human endometrial stromal cells.
- 1992
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Ingår i: Thrombosis Research. - 0049-3848 .- 1879-2472. ; 66:1, s. 75-87
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Cheng, Fang, et al.
(författare)
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Vimentin coordinates fibroblast proliferation and keratinocyte differentiation in wound healing via TGF-beta-Slug signaling
- 2016
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 113:30, s. E4320-E4327
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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