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- Abrahamson, Magnus
(författare)
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Wolfhart Pannenberg och Jesu uppståndelse
- 2004
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Ingår i: Tro & Liv: Tidskrift för kristen tro och förkunnelse. ; 63:3-4, s. 13-21
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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- Ahuja, Sat pal, et al.
(författare)
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Glutathione S-transferase µ(GST) modifies activities of proteases and levels of cystatin C secreted by mouse retinal explants
- 2004
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 45, s. 352-352
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Konferensbidrag (refereegranskat)abstract
- Purpose: In one form of human autosomal recessive retinitis pigmentosa and in retinal degeneration (rd1) mouse, mutation occurs in the genes encoding ß subunit of rod photoreceptor cGMP phosphodiesterase. Therefore, rd1 mutant mouse is an appropriate model for human inherited retinal degeneration studies. Retinal explants are successfully cultured in serum free chemically defined R16 medium to evaluate effects of various rescue factors and retinal conditioned medium (RCM) for secreted molecules like proteases and their inhibitors. Cysteine protease inhibitor cystatin C has recently been identified in rodent neuroretina and RPE. RCM of explants treated with GST were analyzed for proteases and cystatin C to explain, in part, mode of action of GST in protection of degenerating retina. Methods: Postnatal day 2 (PN2) and PN7 control (wt) and rd1 were cultured with (10 ng / ml GST) and without GST in R16 medium, respectively, for 26 and 21 days in vitro (div). Retinal extracts (RE) and RCM were analyzed by fluorometry using casein green fluorescent labeled with BODIPY–FL (Molecular Probes) for total proteases; Z–Phe–Arg–NMec or Z–Arg–Arg–NMec for cysteine proteases and by ELISA for cystatin C, respectively, for levels and secretion of proteases and cystatin C. The protein content of RE was measured. Results: Protein content (µg) of RE from wt and rd1 retinal extracts respectively increased and decreased with age. Cystatin C (ng/ml RCM) content in wt and rd1 RE increased with age (was always higher in wt) up to PN14 and then decreased but was higher than that at PN2. Progressive secretion of cystatin C by PN2 explants was lower than that by PN7 explants; and that by rd1 PN2 and PN7 explants was initially lower up to in vitro age of PN19 and subsequently it was higher than that by wt explants. Secretion of total cystatin C by PN2 and PN7 wt and rd1 explants was similar and was increased by GST. During initial stage of culture total protease activity ({Delta} F / 100 µl RCM) in RCM of rd1 PN2 and PN7 explants was higher and was decreased in GST treated explants. Conclusions: Cystatin C content and secretion by wt RE is always higher and that of proteases is lower than that of rd1. Treatment with GST increases content of cystatin C and consequently decreases that of proteases especially cysteine proteases.
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- Brage, Monica, et al.
(författare)
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Osteoclastogenesis is decreased by cysteine proteinase inhibitors.
- 2004
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Ingår i: Bone. - : Elsevier BV. - 8756-3282 .- 1873-2763. ; 34:3, s. 412-24
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Tidskriftsartikel (refereegranskat)abstract
- The effects of cystatin C and other cysteine proteinase inhibitors on osteoclast formation and differentiation have been investigated. Cystatin C decreased osteoclast formation stimulated by parathyroid hormone (PTH), 1,25(OH)2-vitamin D3 or interleukin-6 (IL-6) (in the presence of its soluble receptor) as assessed by the number of tartrate-resistant acid phosphatase (TRAP+) multinucleated cells in mouse bone marrow cultures. The inhibitory effect was associated with decreased mRNA expression for the calcitonin receptor as well as decreased number of specific binding sites for 125I-calcitonin, and without any effect on the mRNA expression of receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL). Similarly, the cysteine proteinase inhibitors leupeptin, E-64 and benzyloxycarbonyl-Phe-Ala-diazomethane (Z-FA-CHN2) decreased PTH-stimulated formation of TRAP+ multinucleated cells and binding of 125I-calcitonin. A peptidyl derivative synthesized to mimic part of the proteinase-binding site of cystatin C (benzyloxycarbonyl-Arg-Leu-Val-Gly-diazomethane, or Z-RLVG-CHN2) also decreased PTH-stimulated osteoclast formation. In a 9-day culture, addition of cystatin C during the last 5 days was sufficient to cause substantial inhibition of osteoclast formation. Cystatin C-induced decrease of osteoclast formation was associated with enhanced number of F4/80-positive macrophages and increased mRNA expression of the macrophage receptor c-fms in the bone marrow culture. Osteoclast formation in mouse bone marrow cultures as well as in mouse spleen cell cultures, stimulated by macrophage colony-stimulating factor (M-CSF) and RANKL was also decreased by different cysteine proteinase inhibitors. In addition, cystatin C inhibited M-CSF/RANKL induction of calcitonin receptor mRNA in spleen cell cultures. The inhibitory effect by cystatin C in spleen cells was associated with decreased mRNA expression of RANK and the transcription factor NFAT2. It is concluded that cysteine proteinase inhibitors decrease formation of osteoclasts by interfering at a late stage of pre-osteoclast differentiation.
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- Janowski, R, et al.
(författare)
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Domain swapping in N-truncated human cystatin C
- 2004
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 341:1, s. 151-160
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts. (C) 2004 Elsevier Ltd. All rights reserved.
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- Lerner, Ulf H, et al.
(författare)
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Cysteine proteinases in osteoclast function and recruitment
- 2004
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Ingår i: Biological Mechanisms of Tooth Movement and Craniofacial Adaptation. - Boston, Massachusetts, USA : Harvard Society for the Advancement of Orthodontics. - 0963204750 ; , s. 227-227
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 9. |
- Outchkourov, NS, et al.
(författare)
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Engineered multidomain cysteine protease inhibitors yield resistance against western flower thrips (Franklinielia occidentalis) in greenhouse trials
- 2004
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7652 .- 1467-7644. ; 2:5, s. 449-458
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Tidskriftsartikel (refereegranskat)abstract
- Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripiclae), cause very large economic damage on a variety of field and greenhouse crops. In this study, plant resistance against thrips was introduced into transgenic potato plants through the expression of novel, custom-made, multiclomain protease inhibitors. Representative classes of inhibitors of cysteine and aspartic proteases [kininogen domain 3 (K), stefin A (A), cystatin C (C), potato cystatin (P) and equistatin (EIM)] were fused into reading frames consisting of four (K-A-C-P) to five (EIM-K-A-C-P) proteins, and were shown to fold into functional inhibitors in the yeast Pichia pastoris. The multiclomain proteins were expressed in potato and found to be more resistant to degradation by plant proteases than the individual domains. In a time span of 14-16 days, transgenic potato plants expressing EIMKACP and KACP at a similar concentration reduced the number of larvae and adults to less than 20% of the control. Leaf damage on protected plants was minimal. Engineered multiclomain cysteine protease inhibitors thus provide a novel way of controlling western flower thrips in greenhouse and field crops, and open up possibilities for novel insect resistance applications in transgenic crops.
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- Outchkourov, NS, et al.
(författare)
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Specific cysteine protease inhibitors act as deterrents of western flower thrips, Frankliniella occidentalis (Pergande), in transgenic potato
- 2004
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Ingår i: Plant Biotechnology Journal. - : Wiley. - 1467-7652 .- 1467-7644. ; 2:5, s. 439-448
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed.
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