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| 3. |
- Ahuja-Jensen, Poonam, et al.
(författare)
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Low glutathione peroxidase in rdl mouse retina increases oxidative stress and proteases
- 2007
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Ingår i: NeuroReport. - 1473-558X. ; 18:8, s. 797-801
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Tidskriftsartikel (refereegranskat)abstract
- Malondialdehyde, reduced glutathione, glutathione peroxidase, glutathione reductase and cysteine protease cathepsins at postnatal (PN) days 2, 7, 14, 21 and 28 in controls (wt) and the retinal degeneration 1 (rd1) mouse model for retinitis pigmentosa retinas were measured to determine oxidative stress. In PN28 wt and PN2 rd1 retinas, elevated malondialdehyde and low glutathione peroxidase activity indicate higher oxidative load, despite higher reduced glutathione in PN2 rd1 retinas. This is due to physiological exposure to light and retinal vascular/neural restructuring, respectively. Compared with wt retinas, relatively high malondialdehyde at PN2 and cathepsin levels at PN14, 21 and 28 in rd1 retinas indicate that cells of the residual inner retina also contribute to the oxidative stress and retinal degeneration.
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| 4. |
- Ahuja, Sat pal, et al.
(författare)
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Physiopathology of retinal degeneration in rd1 mouse model of retinitis pigmentosa : TGF-Β1, proteinases and oxidative stress mechanisms
- 2009
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Ingår i: Retinal Degeneration: Causes, Diagnosis and Treatment. - 9781607410072 ; , s. 1-41
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Bokkapitel (refereegranskat)abstract
- The rd1 (retinal degeneration) mouse retina shows degeneration homologous to a form of retinitis pigmentosa with a rapid loss of rod photoreceptors and deficiency of retinal blood vessels. Due to Pde6brd1 gene mutation, β subunit of phosphodiesterase (PDE) of rd1 retina has an inactive PDE which elevates cGMP and Ca2+ ions level. In vitro retinal explants provide a system close to the in vivo situation, so both approaches were used to compare the status of oxidative stress, transforming growth factor-β1 (TGF-β1), sialylation, galactosylation of proteoglycans, and different proteinases-endogenous inhibitors systems participating in extracellular matrix (ECM) remodeling/degeneration and programmed cell death (PCD)/apoptosis in wt and rd1 mouse retinas. Proteins and desialylated sulfated glucosaminoglycan parts of proteoglycans in ECM of rd1 retina were, respectively, decreased and increased due to enhanced activities of proteinases. Desialylation increases the susceptibility of cells to phoagocytosis/apoptosis, decreased neurogenesis and faulty guidance cues for synaptogenesis. In vivo activities of total proteinases, matrix metalloproteinase-9 (MMP-9) and cathepsin B were increased in rd1 retina on postnatal day 14 (PN14), -21 and -28, due to relatively lower levels of tissue inhibitor of MMPs (TIMP-1) and cystatin C, respectively. This corresponded with increased in vitro secretion of these proteinases by rd1 retina. Cells including end-feet of Mueller cells in degenerating rd1 retina showed intense immunolabeling for MMP-9, MMP-2/TIMP-1, TIMP-2 and cathepsin B/cystatin C, and proteinases pool was increased by Mueller cells. Intense immunolabeling of ganglion cell (RGC) layer for cathepsin B and of inner-plexiform layer of both PN2/PN7 rd1 and wt retinas indicated importance of cathepsin B in synaptogenesis and PCD of RGC. Increased levels of TGF-β1 in vitro transiently increased the secretion of MMPs and cathepsins activities by wt explants which activate TGF-β1 and remodel the ECM for angiogenesis and ontogenetic PCD. Whereas, lower level of TGF-β1 and persistently higher activities of MMPs and cathepsins in rd1 retinas and conditioned medium, suggested that proteinases degraded TGF-β1 and ECM and caused retinal degeneration. Lower activities of glutathione-S-transferase and glutathione-peroxidase in rd1 retina contribute to oxidative stress which damages membranes and increased the expression, release/secretion of proteinases relative to their endogenous inhibitors. Participation of oxidative stress in rd1 retinal degeneration was further confirmed from the partial protection of rd1 photoreceptors by in vitro and/or in vivo supplementation with glutathione-S-transferase or a combination of antioxidants namely lutein, zeaxanthin, α-lipoic acid and reduced-L-glutathione. Treatment with combination(s) of broad spectrum proteinase inhibitor(s) and antioxidants needs investigation.
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- Ahuja, Sat pal, et al.
(författare)
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rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.
- 2008
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Ingår i: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 49:3, s. 1089-1096
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To compare in vivo levels, spatial localization, and in vitro secretion of cysteine protease cathepsins and cystatin C (cysC) in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa and control (wt) mouse retinas. METHODS: The spatial localization, protein contents, cysC levels and cathepsin-B, -S, and -L activities in wt and rd1 retinas at postnatal (PN) days 2, 7, 14, 21, and 28 were analyzed by immunostaining, spectrophotometry, ELISA, and fluorescence spectrophotometry. The in vitro secretion of cysC and cysteine proteases by PN7 retinal explants into the conditioned medium (RCM) was quantified. RESULTS: The pigment epithelium, photoreceptors, and inner retinal and ganglion cell layers of both wt and rd1 retinas showed cysC and cathepsin-B labeling. CysC immunostaining was extensive in the optic nerve head fibers. The rd1 explants secreted higher amounts of cysteine protease into the RCM. The protein content in wt and rd1 retinal extracts increased up to PN14, then decreased in rd1 but not in wt. In rd1 extracts at PN14 to -28, cathepsin activity was higher and increased with age, but the cysC level was higher and constant. The ratios of cathepsin activity to cysC (cathepsin-L at PN2 and total, -B, and -L at PN14 to -28) were higher in rd1 extracts. CONCLUSIONS: Similar localization of both cathepsin-B and cysC in wt and rd1 retinas along with lower proteins and higher cathepsin activity in rd1 retinal extracts and RCM are consistent with their localization in extracellular matrix and a role in physiopathologic remodeling in wt and rd1 retinas.
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- Ahuja, Sat pal, et al.
(författare)
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rd1 Mouse Retina Shows Imbalance in Cellular Distribution and Levels of TIMP-1/MMP-9, TIMP-2/MMP-2 and Sulfated Glycosaminoglycans.
- 2006
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Ingår i: Ophthalmic Research. - : S. Karger AG. - 1423-0259 .- 0030-3747. ; 38:3, s. 125-136
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Tidskriftsartikel (refereegranskat)abstract
- Background: The rd1 mouse retina displays fast degeneration of photoreceptors resulting in a depletion of almost all rod photoreceptors by postnatal day 21 (PN21). To evaluate the role of proteinases in the pathophysiology of this animal model of retinitis pigmentosa, C3H rd1 and congenic wild-type (wt) mice retinas were analyzed. Material and Methods: The cellular localization and levels of proteins, matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), total sulfated glycosaminoglycans (sGAG) and nature of saccharides in roll and wt retinal extracts were compared. Results: MMP-2/TIMP-2 and MMP-9/TIMP-1 were predominantly localized in the interphotoreceptor matrix (IPM) of both genotypes, but MMP-2/TIMP-2 also appeared in the Muller cell fibers of rd1 retina. In rd1 retinal extracts the levels of total proteins were lower and those of active MMP-9, MMP-2, TIMP-1 and total sGAG were higher than those of wt extracts. Despite an increase in TIMP-1, active MMP-9/MMP-2 were disproportionately elevated in rd1 compared to wt retina. With increasing age, MMPs in wt retinas were decreased but were increased in rd1. The sialylation of proteoglycans in PN2 and PN7 rd1 retinas was lower, and galactosylation was higher than that in wt retinas. Conclusions: MMP-9/ MMP-2 and TIMP-1/TIMP-2 are associated with IPM, possibly after secretion by retinal pigmented epithelial cells. In degenerating rd1 retina, MMP-2/TIMP-2 are associated with the Muller cell fibers, which apparently play a central role in modifying the balance between MMPs and TIMPs. Elevated sGAG and proteolysis due to an imbalance in the levels of TIMPs and active MMP-9/MMP-2 in rd1 retina possibly contribute to retinal degeneration in the rd1 mouse.
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| 7. |
- Alvarez Fernandez, Marcia, et al.
(författare)
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Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile.
- 2005
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 280:18, s. 18221-18228
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Tidskriftsartikel (refereegranskat)abstract
- Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared to its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L, and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg26-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5 and 1.8 Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel -sheet wrapped around a five-turn -helix. The structures reveal differences in the peptidase-interacting regions when compared to other cystatins, providing plausible explanations to the restricted inhibitory specificity of cystatin D for some papain-like peptidases, and its lack of reactivity towards legumain-related enzymes. This is the final, accepted and revised manuscript of this article. Use alternative location to go to the published article. Requires subscription.
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- Bokarewa, Maria, 1963, et al.
(författare)
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Cystatin C binds serum amyloid A, downregulating its cytokine-generating properties
- 2007
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Ingår i: Journal of Rheumatology. - 0315-162X. ; 34:6, s. 1293-1301
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Tidskriftsartikel (refereegranskat)abstract
- Objective. To assess the interaction between cystatin C (CysC) and serum amyloid A protein (SAA). Methods. Levels of CysC and SAA and antibodies against these proteins were assessed in the paired blood and synovial fluid (SF) samples of 90 patients with rheumatoid arthritis (RA). Age and sex matched individuals having normal iohexol clearance (n = 90) and SF following joint trauma (n = 40) were used as controls. In vitro experiments included assessment of interaction between CysC and SAA by ELISA and the influence of CysC on SAA functions. Results. A pilot screening for cystatins C, E, and F in blood and SF of patients with RA found CysC to be by far the predominant extracellular cystatin. Circulating CysC levels were significantly lower in patients with RA compared to the matched controls (0.81 +/- 0.03 vs 1.01 +/- 0.03 mg/l; p = 0.05). These low CysC levels could not be explained by the presence of anti-CysC antibodies in patients with RA. In contrast, concentrations of CysC that accumulated in the inflamed SF were significantly greater in patients with erosive RA (1.66 +/- 0.08 mg/l) compared to nonerosive RA (1.36 +/- 0.06 mg/l; p = 0.003) and controls (1.18 +/- 0.03 mg/l; p = 0.043). In vitro studies showed direct binding of CysC to SAA. CysC/SAA binding impaired proinflammatory effects of SAA, reducing its ability to trigger expression of proinflammatory cytokines. Conclusion. Our study shows a relative deficiency of circulating CysC during systemic inflammation in RA. Physical interaction between CysC and the acute-phase protein SAA (1) provides an explanation for CysC deficiency; and (2) suggests that CysC is regulating inflammatory responses. We hypothesize that decreased systemic CysC levels predispose to accelerated atherosclerosis and development of amyloidosis in patients with RA.
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- Brage, M, et al.
(författare)
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Different cysteine proteinases involved in bone resorption and osteoclast formation.
- 2005
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Ingår i: Calcified tissue international. - : Springer Science and Business Media LLC. - 0171-967X .- 1432-0827. ; 76:6, s. 439-47
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Tidskriftsartikel (refereegranskat)abstract
- Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.
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