Age-related cataract, an opacity of the eye lens, is the leading cause of visual impairment in the elderly, the etiology of which is related to oxidative stress damage. Oxidation of methionine to methionine sulfoxide is a major oxidative stress product that reaches levels as high as 60% in cataract while being essentially absent from clear lenses. Methionine oxidation results in loss of protein function that can be reversed through the action of methionine sulfoxide reductase A (MsrA), which is implicated in oxidative stress protection and is an essential regulator of longevity in species ranging from Escherichia coli to mice. To establish a role for MsrA in lens protection against oxidative stress, we have examined the levels and spatial expression patterns of MsrA in the human lens and have tested the ability of MsrA to protect lens cells directly against oxidative stress. In the present report, we establish that MsrA is present throughout the human lens, where it is likely to defend lens cells and their components against methionine oxidation. We demonstrate that overexpression of MsrA protects lens cells against oxidative stress damage, whereas silencing of the MsrA gene renders lens cells more sensitive to oxidative stress damage. We also provide evidence that MsrA is important for lens cell function in the absence of exogenous stress. Collectively, these data implicate MsrA as a key player in lens cell viability and resistance to oxidative stress, a major factor in the etiology of age-related cataract.

The granulin-epithelin precursor (GEP/PCDGF), a 68-88 kDa secreted glycoprotein, has been shown to be an important growth and survival factor for ovarian cancer cells. Furthermore, GEP expression is a predictor of patient survival in metastatic ovarian cancer cells. Up to this point, however, the molecular mechanisms and clinical relevance of a GEP-mediated prosurvival phenotype remain poorly characterized. We hypothesize that the prosurvival function of GEP is important in ovarian cancer tumor progression and chemoresponse. To explore this hypothesis, we examined the effects of GEP overexpression on migration, invasion and cisplatin (CDDP) chemosensitivity in the ovarian cancer cell line A2780. Full length GEP transfectants demonstrated an increased capacity to migrate and invade their substratum when compared to empty vector controls. In addition, GEP overexpression was associated with CDDP chemoresistance. Finally, GEP overexpression increased tumor formation and protected cells from tumor regression in response to CDDP treatment in vivo. Taken together, these data support a role for GEP in tumor progression and development of drug resistance. ' 2007 Wiley-Liss, Inc.

The present data suggest important roles for both MsrA and -Bs in lens cell viability and oxidative stress protection. The differential tissue distribution and lens expression patterns of these genes, coupled with increased oxidative-stress-induced cell death on their deletion provides evidence that they are important for lens cell function, resistance to oxidative stress, and, potentially, cataractogenesis.

A rat model of low myocardial blood flow was established to test the hypothesis that post-translational changes to proteins of the thin and thick muscle filaments correlate with decreased cardiac contractility. Following 3 days of low blood flow by constriction of the left anterior descending artery, rat hearts demonstrated a reduction in fractional shortening at rest and a relative decline in fractional shortening when challenged with high dose versus low dose dobutamine, reflecting reduced energy reserves. Permeabilized fibers from low blood flow hearts demonstrated a decline in maximum force per cross-section and Ca 2+ sensitivity as compared to their sham operated counterparts. An examination of sarcomeric proteins by twodimensional gel electrophoresis, mass spectrometry, and phospho-specific antibodies provided evidence for Ser23/24 and Ser43/45 phosphorylation of troponin I (TnI). Total TnI phosphorylation was not different between the groups, but Ser23/24 phosphorylation declined with low blood flow, implying an accompanying increase in phosphorylation at other sites of TnI. Affinity chromatography demonstrated that TnI from low blood flow myocardium had reduced relative affinity to Ca 2+ bound troponin C compared to TnI from sham operated hearts, providing a mechanism for reduced Ca 2+ sensitivity of force production in low bloodCorrespondence to: Ozgur Ogut, ogut.ozgur@mayo.edu. Electronic supplementary material The online version of this article

Glutathionylation of intracellular proteins is an established physiological regulator of protein function. In multiple models including ischemia-reperfusion of the heart, increased oxidative stress results in the glutathionylation of sarcomeric actin. We hypothesized that actin glutathionylation may play a role in the multi-factorial change in cardiac muscle contractility observed during this pathophysiological state. Therefore, the functional impact of glutathionylated actin on the interaction with myosin-S1 was examined. Substituting glutathionylated F-actin for unmodified F-actin reduced the maximum actomyosin-S1 ATPase, and this was accompanied by an increase in the activation energy of the steady state ATPase. Measurement of steady state binding did not suggest a large impact of actin glutathionylation on the binding to myosin-S1. However, transient binding and dissociation kinetics determined by stopped-flow methods demonstrated that although actin glutathionylation did not significantly alter the rate constant of myosin-S1 binding, there was a significant decrease in the rate of ATP-induced myosin-S1 detachment in the presence of ADP. These results suggest that actin glutathionylation may play a limited but defined role in the alteration of contractility following oxidative stress to the myocardium, particularly through a decrease in the actomyosin ATPase activity.