| 1. |
- Andersson, Madeleine, et al.
(författare)
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Calcium-dependent proteolysis in rabbit lens epithelium after oxidative stress
- 1998
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Ingår i: Ophthalmic Res. ; 30:3, s. 157-67
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of this study was to examine changes in calcium-dependent proteolytic activity in the lens epithelium from whole rabbit lenses exposed to long-term oxidative stress at near physiological levels. Rabbit lenses, incubated in 50 microM H2O2 for 1 or 24 h, were checked for clarity and morphological changes in the epithelium. Proteolytic activity was measured in the epithelium using a fluorogenic synthetic substrate; N-succinyl-Leu-Tyr-7-amino-4-methylocoumarin, both in the presence and the absence of calcium (1 mM Ca2+ and 5 mM EDTA respectively). The effect on transparency and morphology of the epithelium following a 1-hour incubation in 100 microM H2O2 was also studied. Lenses incubated in 50 microM H2O2 were clear even after 24h. After a 1-hour incubation in 50 microM H2O2 the epithelium of the exposed lens appeared normal. However, after 24 h the epithelium cells appeared swollen and microscopical examination showed extensive intracellular and subepithelial vacuolization. Incubation in 100 microM H2O2 for 1 h caused loss of transparency; vacuole formation, globulization of the superficial lens fibers and death of the epithelial cells. There was a 55% increase in calcium-dependent proteolytic activity after 1 h in 50 microM H2O2, implying a role for the calcium-activated protease calpain in oxidatively induced cataract.
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| 2. |
- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Proteolysis in human lens epithelium as determined by a cell-permeable substrate
- 1998
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Ingår i: Investigative Ophthalmology and Visual Science. ; 40:1, s. 261-264
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS: Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface. RESULTS: After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS: Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.
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| 3. |
- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Proteolysis in human lens epithelium determined by a cell-permeable substrate
- 1999
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Ingår i: Invest Ophthalmol Vis Sci. ; 40:1, s. 261-4
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells. METHODS: Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface. RESULTS: After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin. CONCLUSIONS: Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.
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