| 1. |
- Ahuja, Poonam, et al.
(författare)
-
Decreased glutathione transferase levels in rd1/rd1 mouse retina: Replenishment protects photoreceptors in retinal explants.
- 2005
-
Ingår i: Neuroscience. - : Elsevier BV. - 1873-7544 .- 0306-4522. ; 131:4, s. 935-943
-
Tidskriftsartikel (refereegranskat)abstract
- Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.
|
|
| 2. |
- Ahuja, Sat pal, et al.
(författare)
-
Glutathione S-transferase µ(GST) modifies activities of proteases and levels of cystatin C secreted by mouse retinal explants
- 2004
-
Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 45, s. 352-352
-
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.
|
|
| 3. |
- Ahuja, Sat pal, et al.
(författare)
-
Physiopathology of retinal degeneration in rd1 mouse model of retinitis pigmentosa : TGF-Β1, proteinases and oxidative stress mechanisms
- 2009
-
Ingår i: Retinal Degeneration: Causes, Diagnosis and Treatment. - 9781607410072 ; , s. 1-41
-
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.
|
|
| 4. |
- Ahuja, Sat pal, et al.
(författare)
-
rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.
- 2008
-
Ingår i: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 49:3, s. 1089-1096
-
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.
|
|
| 5. |
- Ahuja, Sat pal, et al.
(författare)
-
rd1 Mouse Retina Shows Imbalance in Cellular Distribution and Levels of TIMP-1/MMP-9, TIMP-2/MMP-2 and Sulfated Glycosaminoglycans.
- 2006
-
Ingår i: Ophthalmic Research. - : S. Karger AG. - 1423-0259 .- 0030-3747. ; 38:3, s. 125-136
-
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.
|
|
| 6. |
- Ahuja, Sat pal, et al.
(författare)
-
Serum-free retinal explant culture system and comparative rescue effects of LEDGF, GST, CNTF, BDNF, NGF, bFGF and antioxidants in the rd1 mouse model of retinitis pigmentosa
- 2009
-
Ingår i: Retinal Degeneration: Causes, Diagnosis and Treatment. - 9781607410072 ; , s. 263-299
-
Bokkapitel (refereegranskat)abstract
- Retinitis pigmentosa is a group of inherited retinal degenerative diseases characterized by the loss of photoreceptors and vision for which no effective treatment is available. Several animal models of retinitis pigmentosa are used to elucidate its pathogenesis and to devise therapies. The retinal degeneration (rd1) mouse is one such animal model in which rod-specific phosphodiesterase (PDE) is inactive due to a mutation in the β-subunit of the Pde gene (Pde6brd1). This mutation leads to increased levels of retinal cyclic guanosine monophosphate (cGMP) and Ca2+ ions and eventually retinal degeneration by increased oxidative stress, activation of poly-(ADP-ribose)-polymerase-1 and proteinases including calpains and caspases. However, diverse overlapping mechanism(s) of cell death have been described. An in vitro retinal explant culture system was developed, as results of studies involving isolated retinal cells and the in vivo models are difficult to interpret. Neonatal and postnatal retinas of wild type (wt) and rd1 mice were cultured successfully in a serum-free medium containing bovine serum albumin. The cultured wt and rd1 retinas respectively developed and degenerated in ways similar to the age-matched in vivo retinas of the two genotypes. This was confirmed from the similar retinal lamination pattern, expression and immunohistochemical localization of opsin, rhodopsin, arrestin, interphotoreceptor retinol-binding protein and calcium-binding protein markers, namely, calbindin, parvalbumin and calretinin in age-matched in vivo and cultured retinas of both genotypes. Horizontal cell fibers and Mueller cells of in vivo retina showed the presence of α-and μ-glutathione-S-transferases (GST). In rd1 mouse retina, GST and glutathione peroxidase (GPx) levels were lower than those in wt and suggested an oxidative stress. The photoreceptor rescue effects of lens epithelium-derived growth factor (LEDGF), α-GST, and μ-GST; and ciliary neurotrophic factor (CNTF), brain-derived growth factor (BDNF), nerve growth factor (NGF), basic fibroblast growth factor (bFGF); and antioxidants (lutein, zeaxanthin, glutathione and α-lipoic acid) were compared after supplementation in a serum-free retina organ culture system. The above combination of antioxidants rescued the rd1 photoreceptors both under in vitro and in vivo conditions. The maximum photoreceptors rescue was by CNTF + BDNF, whereas that by NGF + bFGF was intermediate. CNTF, BDNF, NGF, and bFGF individually rescued the photoreceptors, but their effect was less than those of their combinations. LEDGF and GST supplementation in vitro delayed rod photoreceptor degeneration in postnatal day-2 (PN2) and PN7 rd1 explants cultured in vitro for 26 and 21 days, respectively, possibly by reversing the oxidative stress. The latter was confirmed from the lower levels of GPx and from the rescue of rd1 photoreceptors by in vitro and in vivo supplementation with LEDGF or the combination of antioxidants. Individual antioxidants were ineffective in rescuing the photoreceptors under in vivo and in vitro conditions.
|
|
| 7. |
- Ahuja, Sanjay, et al.
(författare)
-
Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein I domains in Escherichia coli
- 2006
-
Ingår i: Malaria Journal. - : Springer Science and Business Media LLC. - 1475-2875. ; 5
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL ( Duffy binding like), CIDR ( cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. Methods: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. Results: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1 alpha (46.8%) > CIDR2 beta (42.9%) > DBL2-4 gamma (31.7%) > DBL2 beta + C2 (30.6%) > DBL1 alpha (24.9%) > DBL2-7 epsilon (23.1%) > DBL2-5 delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. Conclusion: The acidic terminal segment ( ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.
|
|
| 8. |
|
|
