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| 2. |
- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Acute Effects of Indomethacin on Mitochondrial Function and Glutathione Levels in the Mouse Lens in Organ Culture
- 2003
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 44:5, s. 3500-
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Konferensbidrag (refereegranskat)abstract
- Purpose, Experiments were carried out to study the effects of indomethacin on markers for oxidative damage and mitochondrial function in the intact mouse lens. Methods, Mouse lenses were incubated with fluorogenic indicator dyes prior to administration of 50 {micro}M indomethacin. The response was monitored, in real time, for several hours. Mitochondrial depolarization was followed by preloading the lens with Rhodamine 123 (10 {micro}g/ml). Peroxide production was studied in lenses loaded with 50{micro}M DCF-DA and superoxide levels with 10 {micro}M hydroethidium. Glutathione levels were assayed with 50 {micro}M monochlorobimane following short-term administration of indomethacin. Results, No significant changes in the production of peroxide or superoxide were found up to 3 hours after administration of indomethacin. The level of reduced glutathione was reduced by approximately 10 percent 3 h after drug administration. There was a significant increase (20%) of mitochondrial depolarization, compared to control lenses, one hour following indomethacin administration. Conclusions, The frequent use of nonsteroidal anti-inflammatory drugs in ophthalmology necessitates a close examination of effects besides the anti-inflammatory ones. This study points to relatively rapid effects on mitochondrial function and glutathione levels in the cultured mouse lens. The possible relation between these changes and the development of cataract remains to be studied.
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| 3. |
- Karlsson, Jan-Olof, 1944, et al.
(författare)
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Inhibition Of Glycogen Synthase Kinase (gsk-3) Affects Markers Of Oxidative Stress And Attenuates Apoptosis In Human Lens Epithelial Cells
- 2004
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 45:5, s. 3508-
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Konferensbidrag (refereegranskat)abstract
- Purpose: GSK-3, an evolutionary conserved S/T kinase which regulates cell fate determination in diverse organisms have been implicated in the formation of amyloid b-peptides and the phosphorylation of tau and catenin. Inhibition of GSK-3 can be obtained via the structurally unrelated substances lithum or Kenpaullone. The lens and the lens epithelial cells are excellent models to study the role of this enzyme. Methods: Confluent human lens epithelial cells (HLEC) were exposed to the GSK-3 inhibitors lithium (2 mM) or Kenpaullone (2 {micro}M) for times upp to 24h. The cells were, before or after treatment, placed in medium containing fluorogenic indicators of oxidative damage. DCFH-DA was used to assay peroxides, mitochondrial function was evaluated with Rhodamine 123, monochlorobimane was used to assay intracellular glutathione (GSH) levels, Proteolytic activities were assayed, on line, with cell-permeable fluorogenic substrates.Proteasome and calpain activities were assayed with LLVY-AMC, Cathepsin B with RR-AMC or FR-AMC. Metalloproteases were assayed with AAF-AMC. Caspase-3, 8 and 9 were assayed in cell extracts with DEVD-, IETD- or LEHD-AMC, respectively. Results: The mitochondrial membrane potential and the level of GSH increased by 10% after treatment with Li or Kenpaullone for 24h. No change was observed for peroxide production. The basal (low) level of caspase-3 activity was decreased by 20%. No significant effects were found concerning caspase-8 or 9 activities. No effect was observed on the activity of calpain, the proteasome, metalloproteases and cathepsin D/E activity. Conclusions:Inhibition of GSK-3 may protect against oxidative damage and attenuate apoptosis in HLEC. No changes of the other major proteolytic systems in the cell were detected. These data may be important for the interpretation of Wnt signaling and cell growth in HLEC as well as for the formation of amyloid in the lens.
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| 4. |
- Petersen, Anne, 1962, et al.
(författare)
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A new model for assessing proteolysis in the intact mouse lens in organ culture
- 2004
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Ingår i: Ophthalmic Res. - 1423-0259. ; 36:1, s. 25-30
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To develop a new method to investigate proteolysis in the intact lens in organ culture. METHODS: Intact mouse lenses were assayed at regular intervals for proteolytic activity using fluorogenic peptide substrates +/- addition of ionomycin. Specific inhibitors were used to determine the activity of calpains, the proteasome and acid lysosomal enzymes. RESULTS: Significant levels of proteolytic activity were present in the intact lens. Proteolysis was stimulated by ionomycin. Preincubation with an inhibitor to the proteasome significantly decreased proteolysis whereas inhibitors of calpain and acid lysosomal enzymes did not. CONCLUSION: This study indicates that in the intact mouse lens in culture, the proteasome is an important protease. Its activity is at least partially regulated by calcium.
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- Petersen, Anne, 1962, et al.
(författare)
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COX-inhibitors and ASA; influence on oxidative stress in cultured mouse lens
- 2004
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 45:5, s. 395-
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Konferensbidrag (refereegranskat)abstract
- Purpose: Several substances such as ASA and COX-inhibitors have been attributed antioxidant properties. The main objectives of this study were to examine possible protection against oxidative damage in cultured mouse lens by ASA, specific COX-2 as well as unspecific COX-1-2 inhibitors. Methods: Intact mouse lenses were exposed to acetylsalicylic acid (0.03 mM), celecoxib (0.5 {micro}M), indomethacin (0.5 {micro}M), diclofenac (0.5 {micro}M), and/or H2O2 (100 {micro}M) for 8 days. H2O2 were administered as a single dose each day. Lens transparency was examined every day by light microscopy. The intact lenses were assayed at regular intervals during 6 h for proteolytic activity using the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC (LLVY-AMC) (50 {micro}M) suitable for the calpain and proteasome proteolytic systems. Prior to the oxidative stress experiment, dose-response curves had been performed in a similar manner using doses ranging between 0.5-50 {micro}M for COX-inhibitors, 0.03-3 mM for ASA and 10-100 {micro}M for H2O2. Results: Dose-response experiments showed no effect by COX-inhibitors at 0.5 {micro}M or ASA at 0.03 mM on proteolysis or lens transparency compared to control. Hence, these doses of drugs were used to look at possible protective effects in oxidative stress experiments. Opacification of lenses exposed to a single dose of 100 {micro}M H2O2 each day appeared at day 5. Proteolytic activity of the calpain - proteasome systems in lenses exposed to H2O2 in the presence of ASA, COX-2 or COX-1-2 inhibitors did not differ significantly from lenses exposed to H2O2. No protection against oxidative stress by these substances could be detected with regard to change in lens transparency. Conclusions: The protective effect of ASA, COX-2 and COX-1-2 inhibitors against oxidative damage in the lens may be limited. The mechanisms through which these substances exert their actions with respect to oxidative damage require further studies.
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| 6. |
- Petersen, Anne, 1962, et al.
(författare)
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Effect of NSAIDs on Proteolytic Activity and Morphology of the Mouse Lens in Organ Culture
- 2003
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Ingår i: Invest. Ophthalmol. Vis. Sci.. ; 44:5, s. 3499-
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Konferensbidrag (refereegranskat)abstract
- Purpose: To study the influence of nonsteroidal anti-inflammatory drugs (NSAIDs) on proteolytic activity in the mouse lens in organ culture and to examine possible morphological changes indicative of cataract induction. Methods: Mouse lenses were incubated in Earle's balanced salt solution for 24 hours prior to addition of indomethacin or diclofenac at 0.5, 5, or 50 {micro}M. The lenses were exposed to the NSAIDs from 1 to 12 days. Clarity of the lenses was studied every day. The proteolytic assay was performed on a microtiter plate using a fluorogenic substrate, Suc-Leu-Leu-Val-Tyr-AMC (LLVY-AMC) (50 {micro}M) suitable for the calpain and proteasome proteolytic systems. The preparation was assayed at regular intervals for degradation of the substrate during 24 hours. Morphology of the lenses was studied after the experiment by light microscopy. Results: Opacification of the lenses appeared at day 8 at 50{micro}M and at day 12 at 5{micro}M NSAID concentration for indomethacin as well as for diclofenac. However, the indomethacin-incubated lenses expressed a more severe turbidity than diclofenac-incubated ones. Following an initial decrease in proteolysis (day 1-3), incubation with indomethacin resulted in increased proteolytic activity (4-12 days) of calpain and/or the proteasome. Lenses exposed to diclofenac exhibited increased degradation of LLVY as compared to control lenses during the whole incubation period. Morphological examination of the lenses exposed to NSAIDs showed swelling of outer cortical fibres at 0.5 {micro}M. Higher concentrations presented major morphological changes, such as disrupted epithelial cells and swollen, partly globulized cortical fibres. Conclusion: The role of NSAIDs in cataract formation is still unclear; previous data has indicated a cataractogenic as well as a potential protective effect of NSAIDs against cataract formation. Our study demonstrates proteolytic disturbances in the calpain and proteasome system after NSAID administration and also morphological changes. Further studies are required to evaluate NSAID as a potential cataractogenic risk factor.
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| 8. |
- Zetterberg, Madeleine, 1969, et al.
(författare)
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Proteasome activity in human lens nuclei and correlation with age, gender and severity of cataract
- 2003
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Ingår i: Curr Eye Res. ; 27:1, s. 45-53
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: The aim of this study was to measure proteasome activity in human lens nuclei resulting from cataract surgery and in different regions of donor lenses. METHODS: The chymotrypsin-like, the trypsin-like and the peptidylglutamyl-peptide hydrolysing activities of the proteasome were studied using synthetic flourogenic substrates. RESULTS: Proteasome activity did not show any correlation with age of the patients or with gender. Increased opacification of the lens nucleus, as estimated prior to surgery using a 4-grade scale, was significantly correlated with decreased activity of all peptidase activities in the insoluble fraction. In the donor lenses, all peptidase activities were highest in the epithelium and decreased rapidly towards the nucleus. CONCLUSIONS: The present study demonstrates that proteasome activity is preserved in the nucleus of lenses from elderly individuals, although a decrease can be seen with cataract formation. This finding may be of importance for elucidating the mechanism behind the formation of nuclear cataract.
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