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      Decreased carbohydrate metabolism enzyme activities in the glaucomatous trabecular meshwork

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          Abstract

          Purpose

          To determine whether activity of carbohydrate metabolism enzymes (aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls.

          Methods

          Tissue specimens were obtained from trabeculectomy (n=45 open angle glaucoma; Caucasian, average age 61±8 years of age of both genders) and from cadaver eyes (n=15 control and n=5 glaucoma; Caucasian, average age 63±4 years of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically at 240 nm absorbance using reaction of 3-phosphoglycerate with hydrazine to form hydrazone. Pyruvate kinase activity was measured by coupling lactate dehydrogenase with NADPH and pyruvate absorbance was measured at 340 nm. Isocitrate dehydrogenase activity was measured using reduction of NADP to NADPH at the characteristic absorbance at 340 nm. Malate dehydrogenase catalyzes the interconversion of L-malate and oxaloacetate using NADP as a coenzyme, quantified by its absorbance at 340 nm.

          Results

          Aldolase, pyruvate kinase, isocitrate dehydrogenase, and malate dehyrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50, 25, and 69 percent, respectively. SDS–PAGE analysis suggests the presence of 4-hydorxynonenal (HNE) modified isocitrate dehydrogenase protein in the glaucomatous TM tissue compared to controls.

          Conclusions

          Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein. Lipid peroxidation product modification of aldolase, pyruvate kinase, and isocitrate dehydrogenase serves as a likely reason for the reduction of enzyme activity.

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          Most cited references 15

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          Nonlinear fitting method for determining local false discovery rates from decoy database searches.

          False discovery rate (FDR) analyses of protein and peptide identification results using decoy database searching conventionally report aggregate or global FDRs for a whole set of identifications, which are often not very informative about the error rates of individual members in the set. We describe a nonlinear curve fitting method for calculating the local FDR, which estimates the chance that an individual protein (or peptide) is incorrect, and present a simple tool that implements this analysis. The goal of this method is to offer a simple extension to the now commonplace decoy database searching, providing additional valuable information.
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            Mitochondrial complex I defect induces ROS release and degeneration in trabecular meshwork cells of POAG patients: protection by antioxidants.

            There is growing evidence that oxidative stress contributes to the progression of primary open-angle glaucoma (POAG), a leading cause of irreversible blindness worldwide. The authors provide evidence that mitochondrial dysfunction is a possible mechanism for the loss of trabecular meshwork (TM) cells in persons with POAG. TM from patients with POAG (GTM) and age-matched subjects without disease (NTM) were obtained by standard surgical trabeculectomy. Primary TM cultures were treated with one of the following mitochondrial respiratory chain inhibitors: rotenone (ROT, complex I inhibitor), thenoyltrifluoroacetone (TTFA, complex II inhibitor), myxothiazol or antimycin A (MYX, AM-complex III inhibitors); mitochondrial permeability transition (MPT) inhibitor cyclosporine A (CsA); and antioxidants vitamin E (Vit E) or N-acetylcysteine (NAC). Mitochondrial function was determined by changes in mitochondrial membrane potential (DeltaPsim) and adenosine triphosphate (ATP) production with the fluorescent probes 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively. Reactive oxygen species (ROS) level, determined by H(2)-DCF-DA, and cell death, measured by lactate dehydrogenase activity and Annexin V-FITC labeling, were also examined. GTM cells have higher endogenous ROS levels, lower ATP levels, and decreased Delta Psi m and they are more sensitive to mitochondrial complex I inhibition than their normal counterparts. ROT induces a further increase in ROS production, the release of cytochrome c, and decreases in ATP level and Delta Psi m in GTM cells, eventually leading to apoptosis. Complex II and III inhibition had little effect on the cells. Antioxidants protect against ROT-induced death by inhibiting ROS generation and cytochrome c release. The authors propose that a mitochondrial complex I defect is associated with the degeneration of TM cells in patients with POAG, and antioxidants and MPT inhibitors can reduce the progression of this condition.
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              Oxidative stress is an early event in hydrostatic pressure induced retinal ganglion cell damage.

              To determine whether oxidative adduct formation or heme oxygenase-1 (HO-1) expression are altered in retinal ganglion cell (RGC) cultures exposed to elevated hydrostatic pressure and in a mouse model of glaucoma. Cultured RGC-5 cells were subjected to 0, 30, 60, or 100 mm Hg hydrostatic pressure for 2 hours, and the cells were harvested. Parallel experiments examined the recovery from this stress, the effect of direct 4-hydroxy-2-nonenal (HNE) treatment, and the effect of pretreatment with resveratrol or quercetin. Mice were anesthetized and intraocular pressure was increased to 30, 60, or 100 mm Hg for 1 hour; then the retinas were harvested. HNE adduct formation and HO-1 expression were assessed by immunocytochemistry and immunoblotting. Increases of HNE-protein adducts (up to 5-fold) and HO-1 expression (up to 2.5 fold) in pressure-treated RGC-5 cells were dose dependent. During recovery experiments, HNE-protein adducts continued to increase for up to 10 hours; in contrast, HO-1 expression decreased immediately. HNE, at a concentration as low as 5 muM, led to neurotoxicity in RGC-5 cells. HNE adducts and HO-1 expression increased in the mouse retina and optic nerve after acute IOP elevation up to 5.5-fold and 2-fold, respectively. Antioxidant treatment reduced the oxidative stress level in pressure-treated RGC-5 cells. This study demonstrates that oxidative stress is an early event in hydrostatic pressure/IOP-induced neuronal damage. These findings support the view that oxidative damage contributes early to glaucomatous optic neuropathy.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2010
                10 July 2010
                : 16
                : 1286-1291
                Affiliations
                [1 ]Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL
                [2 ]Miami Veterans Affairs Health Care System, Miami, FL
                [3 ]Mundorf Eye Center, Charlotte, NC
                [4 ]Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH
                Author notes
                Correspondence to: Anna K. Junk, 900 NW 17th Street, Miami, FL, 33136; Phone: (305) 326-6447; FAX: (305) 326-6432; email: ajunk@ 123456med.miami.edu
                Article
                142 2010MOLVIS0148
                2904041
                20664702
                Copyright © 2010 Molecular Vision.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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                Vision sciences

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