| 1. |
- 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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| 2. |
- Kling-Petersen, Anne, 1962
(författare)
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Oxidative stress and proteolysis in the lens. Effects of anti-inflammatory drugs
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxidative stress has been implied in formation of age-related cataract. The objectives of this thesis were to study redox regulation of proteolysis and oxidative/antioxidative effects of anti-inflammatory substances in human lens epithelial cells (HLEC) and intact mouse lenses.Human lens epithelial cells and intact mouse lenses were exposed to the non-steroid anti-inflammatory substances (NSAIDs) indomethacin, diclofenac, celecoxib and acetylsalicylic acid (ASA) as well as to the glucocorticoid dexamethasone. HLEC were oxidatively stressed by H2O2. Apoptosis was studied by Hoechst staining of cell nuclei and caspase-3 activity. Cells were assayed for changes in superoxide production using dihydroethidium, for alterations in peroxide production using dichlorofluorescin diacetate and for glutathione variations using monochlorobimane. Mitochondrial depolarisation was measured with the potential-sensitive dye JC-1. Morphology was studied by transmission electron microscopy (TEM). Proteolytic activity of calpain, the proteasome and acid lysosomal proteases was measured using fluorogenic substrates. Low concentrations of NSAIDs/ASA protected against H2O2-induced apoptosis in HLEC whereas higher concentrations were toxic. Low concentrations of NSAIDs/ASA reduced superoxide and peroxide production as well as GSH depletion in oxidatively stressed HLEC. No protection of NSAIDs/ASA against H2O2-induced mitochondrial membrane potential depolarisation could be seen. Dexamethasone significantly increased apoptosis, but had no effect on superoxide production, GSH-levels or mitochondrial membrane potential. Only the highest concentration of dexamethasone (100 µM) showed an increase in peroxide production. TEM revealed multilayering of cells, vacuole formation and appearance of electron-dense multivesicular bodies in HLEC exposed to dexamethasone. A new method for measuring proteolysis in intact mouse lenses was established. Proteolytic activity was stimulated by increased intracellular calcium concentrations, as viewed by addition of ionomycin. The proteasome inhibitor lactacystin significantly decreased proteolysis whereas inhibitors of calpain and acid lysosmal enzymes did not. All three main peptidase activities of the proteasome in HLEC lysate were affected by incubation with reduced or oxidized glutathione (GSH, GSSG). The chymotrypsin-like and peptidylglutamyl peptidase-like activities responded similarly with stimulation upon addition of GSH, whereas the trypsin-like activity was inhibited. Addition of GSSG caused inhibition of all three peptidase activities, but simultaneous incubation with DTT reversed the inhibitory effect of GSSG. Oxidative stress induced by H2O2 significantly decreased the main peptidase activities of the proteasome in cultured HLEC. Intact mouse lenses incubated with H2O2 showed trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH-levels.In conclusion, NSAIDs/ASA protects against apoptosis in oxidatively stressed HLEC by antioxidative actions. Steroid-exposed HLEC exhibit multilayering of cultured cells and increased apoptosis indicating disturbed differentiation/proliferation/migration as probable mechanisms behind steroid-induced cataract. Proteolysis in lens epithelial cells is strictly regulated by intracellular redox status.
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| 3. |
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| 4. |
- Li, J. Y., et al.
(författare)
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Distribution of Rab3a in rat nervous system: comparison with other synaptic vesicle proteins and neuropeptides
- 1996
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Ingår i: Brain Research. ; 706:1, s. 103-112
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Tidskriftsartikel (refereegranskat)abstract
- In the present study we have investigated the distribution of Rab3a in rat peripheral nervous system and compared it with the distribution of other synaptic vesicle proteins (synaptophysin, synapsin I), neuropeptides (CGRP, SP, NPY) and tyrosine hydroxylase (TH). Rab3a immunoreactivity (-IR) was always colocalized with synaptophysin-IR and synapsin I-IR in nerve terminals of the spinal cord and peripheral nerve endings. In many cases, Rab3a-IR was also present in the same axons and terminals as peptides. In crushed sciatic nerve axons, Rab3a was colocalized, proximal to the crush, with synaptophysin-IR, synapsin I-IR, CGRP-IR, and TH-IR, but only partially co-localized with NPY-IR and SP-IR. In the area distal to the crush, Rab3a-IR was very weakly positive in a few thin axons, while larger amount of synaptophysin, CGRP, NPY and SP immunoreactivities were detected. The subcellular distribution of peptides and Rab3a differed in that peptides were observed mainly in large granular structures, while Rab3a-IR was observed mainly as diffuse, finely granular immunoreactivity, in addition to a few exceptional large granules present in some axons. The results demonstrate that Rab3a is widely distributed in different types of neurons, i.e. motor, sensory, autonomic adrenergic and cholinergic neurons, and colocalized with other synaptic vesicle proteins, suggesting that Rab3a may play an essential role in neuronal function. Furthermore, Rab3a is present in many peptide containing axons and terminals, but with an apparently different subcellular distribution, being affiliated mostly with small synaptic vesicles and only occasionally with large vesicles, that may represent peptide contained vesicles.
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| 5. |
- Li, Jia-Yi, et al.
(författare)
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GAP 43-like immunoreactivity in normal adult rat sciatic nerve, spinal cord, and motoneurons: axonal transport and effect of spinal cord transection
- 1993
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Ingår i: Neuroscience. - 0306-4522. ; 57:3, s. 759-76
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Tidskriftsartikel (refereegranskat)abstract
- Using immunofluorescence and cytofluorimetric scanning techniques in the rat, the fast anterograde and retrograde axonal transport of growth-associated protein-43-like immunoreactivity in normal sciatic nerves, and after spinal cord transection in the lower thoracic region, were investigated. Spinal roots and motor endplates in the peroneal muscles were also studied. For comparison, anti-synaptophysin (p38) was used. In intact adult animals, the amounts of immunoreactive growth-associated protein-43 increased linearly, both proximally and distally to the crush site, between 1 and 24 h after crushing the sciatic nerve. The accumulations were present in thick as well as in thin axons. Distal accumulations in the sciatic nerve were about 40-60% of the proximal amounts, indicating a recycling of organelles with growth-associated protein-43-like immunoreactivity. During the week after spinal cord transection, no clear changes were observed; the anterograde transport of growth-associated protein-43-like immunoreactivity showed a tendency to decrease at day 1 and then a tendency to increase, reaching 120% of control at seven days (not significant). Transported p38-like immunoreactivity showed similar but smaller changes. In the lumbar spinal cord gray matter many nerve terminals with growth-associated protein-43-like immunoreactivity were seen in intact animals. After spinal transection, these terminals gradually decreased, suggesting that they belonged to descending pathways. However, p38-positive terminals were not obviously decreased. After crushing ventral and dorsal roots, accumulations of pf growth-associated protein-43-like immunoreactivity were present in thick axons in the ventral roots and in thin to medium-sized axons in the dorsal roots. In peroneal muscles, growth-associated protein-43-like immunoreactivity was present in some (but not all) motor endplates in all groups. These results indicate that: (i) growth-associated protein-43 is normally present in nerve terminals of many descending projections of the spinal cord; (ii) growth-associated protein-43-like immunoreactivity is expressed and bidirectionally transported in neurons (motor as well as sensory) of normal sciatic nerves; (iii) growth-associated protein-43-like immunoreactivity is present in some adult motor endplates; and (iv) inhibited supraspinal input causes minor, if any, alterations--paralleled by p38--in axonal transport of growth-associated protein-43-like immunoreactivity.
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| 6. |
- Li, Jia-Yi, et al.
(författare)
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Influence of spinal cord transection on the presence and axonal transport of CGRP-, chromogranin A-, VIP-, synapsin I-, and synaptophysin-like immunoreactivities in rat motor nerve
- 1992
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Ingår i: J Neurobiol. - 0022-3034. ; 23:8, s. 1094-110
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Tidskriftsartikel (refereegranskat)abstract
- Using immunofluorescence and cytofluorimetric scanning (CFS), we investigated the short-term (1-7 days) influence of lower thoracic spinal cord transection on lumbar motor neurons. The content of calcitonin gene-related peptide- (CGRP) like immunoreactivity (LI), chromogranin A (Chr A)-LI, vasoactive intestinal polypeptide (VIP)-LI, Syn I-LI, and synaptophysin (p38)-LI in motor perikarya, and the anterograde and retrograde axonal transport of these substances in the sciatic nerve, were studied in nerve crush (6 h) experiments. During the week after transection, CGRP-LI in perikarya decreased, whereas Chr A-LI increased. VIP-LI, co-localized with Chr A-LI in motor perikarya, did not change after transection. The antero- and retrograde transport of CGRP-LI in the sciatic nerve, occurring in both motor and sensory axons, appeared unchanged in cytofluorimetric scanning (CFS) graphs, but the microscopical picture clearly showed that large motor axons had a decreased content of CGRP-LI at 3 and 7 days posttransection, whereas thinner axons were unchanged in fluorescence intensity. The anterograde transport of Chr A-LI, present in both motor and postganglionic adrenergic axons, was decreased 1 and 3 days after lesion, but returned to control by day 7. There was a marked decrease in anterograde transport of VIP-LI, present mainly in postganglionic sympathetic axons, at day 3, but at 7 days transport was normal. The amounts of transported p38, the synaptic vesicle marker, were in the normal range during the whole period. Syn I-LI accumulation anterogradely was somewhat decreased at 3 and 7 days posttransection, and at 1 day the retrograde accumulation was significantly increased. The results suggest that removal of supraspinal input to intact lower motor neurons causes alterations in metabolism and axonal transport of organelle-associated substances, partly probably related to the complex pattern of transmitter leakage from degenerating, descending nerve terminals. These alterations appear to take place also in postganglionic sympathetic neurons in the sciatic nerve, that originate in the lumbar sympathetic chain.
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