Acid phosphatase and lipid peroxidation in human cataractous lens epithelium

The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract

The 5HT 1a Receptor Agonist 8-Oh DPAT Induces Protection from Lipofuscin Accumulation and Oxidative Stress in the Retinal Pigment Epithelium

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagyderived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 mM H 2O 2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H2O2-treated cells compared to controls and protected against H 2O 2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in th

8-OH DPAT reduces lipofuscin accumulation in cultured RPE cells.

<p><b>A</b> - The effect of different concentrations of 8-OH DPAT on lipofuscin accumulation from autophagy in human ARPE19 cells. Cells were maintained in basal medium and received 10 µM 8-OH DPAT every two days for four weeks. <b>B</b> - Autofluorescent intensity of ARPE19 cells treated with 10 µM 8-OH DPAT every two days for up to 4 weeks. Control cells received vehicle alone. <b>C</b> - Fluorescent micrographs of ARPE19 cells as described in B showing reduced lipofuscin granules in 8-OH DPAT treated cells compared to controls. <b>D</b> - Autofluorescent intensity of ARPE19 cells fed POS treated with 10 µM 8-OH DPAT every two days for up to 14 days. Control cells received POS and saline vehicle alone. <b>E</b> - Fluorescent micrographs of ARPE19 cells described in D showing reduced lipofuscin granule accumulation in 8-OH DPAT treated cells fed POS compared to untreated controls. Fluorescence intensity was determined by flow cytometric analysis. Data represent the mean of three experiments. Bar marker is 50 µM.</p

ERG a-wave and b-wave amplitudes and retinal thickness measured in AAV-ribozyme SOD2 knockdown eyes and control eyes treated with 8-OH DPAT.

<p>Eyes received injection of AAV-<i>SOD2</i> ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. ERGs were obtained at 1 and 4 months following virus injection: <b>A & B</b> - ERG wave-forms from mice treated with AAV-mCherry (<b>A</b>) or AAV-VMD2-<i>SOD2</i> Rz (<b>B</b>). Simultaneous full-field ERG measurements were recorded in dark-adapted mice four months after injection with AAV. The scale units on the ordinate are 100 microvolts. The graphs show wave forms for mice treated with saline, blue lines, low dose (0.5 mg/kg) 8-HO-DPAT, red lines, or high dose (5 mg/kg) 8-HO-DPAT, green lines. <b>C</b> – a-wave; <b>D</b> – b-wave. <b>E</b> - ONL thickness measured by SD-OCT at 1 and 4 months. Untreated wild type animals acted as the baseline control. (10 animals per group, P≤0.01 for all doses and time points).</p

8-OH DPAT reduces superoxide anion generation and increases antioxidant capacity in cultured RPE cells.

<p>Cells were exposed to H₂O₂ (200 µM) for 1 hour and either pre-or post treated with 8-OH DPAT (10 µM) for 24 hours. In the case of pretreatment all measurements were made 24 hr after H₂O₂ and for post treatment 8-OH DPAT was added immediately following H₂O₂ exposure. <b>A</b> - Superoxide generation was measured using FACS analysis following staining with MitoSOX and results are expressed as the mean fluorescence intensity. <b>B</b> - SOD2 levels were determined by Western blot analysis. <b>C</b> - The ratio of reduced glutathione (GSH) to the oxidized form of glutathione (GSSG) was measured by ELISA. Data represent the mean of three experiments.</p

8-OH DPAT decreases oxidative stress in the RPE of SOD2 knockdown eyes of mice.

<p>Eyes received injection of AAV-<i>SOD2</i> ribozyme or AAV-mCherry and animals received subcutaneous 8-OH DPAT or saline control for up to 4 months. Sections were stained for 8-hydroxydeoxyguanosine (8OHdG) (green). <b>A</b> – Representative sections of 8OHdG expression in the RPE of control and SOD2 eyes receiving different concentrations of 8-OH DPAT. <b>B</b> - Graph shows quantitation of 8OHdG fluorescence in the RPE layer.</p

Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation

A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3′ untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression