Modulation of the DNA-binding activity of Saccharomyces cerevisiae MSH2–MSH6 complex by the high-mobility group protein NHP6A, in vitro

DNA mismatch repair corrects mispaired bases and small insertions/deletions in DNA. In eukaryotes, the mismatch repair complex MSH2–MSH6 binds to mispairs with only slightly higher affinity than to fully paired DNA in vitro. Recently, the high-mobility group box1 protein, (HMGB1), has been shown to stimulate the mismatch repair reaction in vitro. In yeast, the closest homologs of HMGB1 are NHP6A and NHP6B. These proteins have been shown to be required for genome stability maintenance and mutagenesis control. In this work, we show that MSH2–MSH6 and NHP6A modulate their binding to DNA in vitro. Binding of the yeast MSH2–MSH6 to homoduplex regions of DNA significantly stimulates the loading of NHP6A. Upon binding of NHP6A to DNA, MSH2–MSH6 is excluded from binding unless a mismatch is present. A DNA binding-impaired MSH2–MSH6F337A significantly reduced the loading of NHP6A to DNA, suggesting that MSH2–MSH6 binding is a requisite for NHP6A loading. MSH2–MSH6 and NHP6A form a stable complex, which is responsive to ATP on mismatched substrates. These results suggest that MSH2–MSH6 binding to homoduplex regions of DNA recruits NHP6A, which then prevents further binding of MSH2–MSH6 to these sites unless a mismatch is present

Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses

Hypertension is a disorder affecting millions worldwide, and is a leading cause of death and debilitation in the United States. It is widely accepted that during hypertension and other cardiovascular diseases the vasculature exhibits endothelial dysfunction; a deficit in the relaxatory ability of the vessel, attributed to a lack of nitric oxide (NO) bioavailability. Recently, the one electron redox variant of NO, nitroxyl anion (NO-) has emerged as an endothelium-derived relaxing factor (EDRF) and a candidate for endothelium-derived hyperpolarizing factor (EDRF). NO- is thought to exist protonated (HNO) in vivo, which would make this species more resistant to scavenging. However, no studies have investigated the role of this redox species during hypertension, and whether the vasculature loses the ability to relax to HNO. Thus, we hypothesize that aorta from angiotensin II (AngII)-hypertensive mice will exhibit a preserved relaxation response to Angeli's Salt, an HNO donor. Male C57B16 mice, aged 12-14 weeks were implanted with mini-osmotic pumps containing AngII (90 ng/min, 14 days plus high salt chow) or sham surgery. Aorta were excised, cleaned and used to perform functional studies in a myograph. We found that aorta from AngII-hypertensive mice exhibited a significant endothelial dysfunction as demonstrated by a decrease in acetylcholine (ACh)-mediated relaxation. However, vessels from hypertensive mice exhibited a preserved response to Angeli's Salt (AS), the HNO donor. To confirm that relaxation responses to HNO were maintained, concentration response curves (CRCs) to ACh were performed in the presence of scavengers to both NO and HNO (carboxy-PTIO and L-cys, resp.). We found that ACh-mediated relaxation responses were significantly decreased in aorta from sham and almost completely abolished in aorta from AngII-treated mice. Vessels incubated with L-cys exhibited a modest decrease in ACh-mediated relaxations responses. These data demonstrate that aorta from AngII-treated hypertensive mice exhibit a preserved relaxation response to AS, an HNO donor, regardless of a significant endothelial dysfunction. (C) 2011 Elsevier Ltd. All rights reserved

The antioxidant requirement for plasma membrane repair in skeletal muscle.

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo

HMG-CoA Reductase Inhibitors (Statin) Prevents Retinal Neovascularization in a Model of Oxygen-Induced Retinopathy

PURPOSE. Retinal neovascularization (RNV) is a primary cause of blindness and involves the dysfunction of retinal capillaries. Recent studies have emphasized the beneficial effects of inhibitors of HMG-CoA reductase (statins) in preventing vascular dysfunction. In the present study, the authors characterized the therapeutic effects of statins on RNV. METHODS. Statin treatment (10 mg/kg/d fluvastatin) was tested in a mouse model of oxygen-induced retinopathy. Morphometric analysis was conducted to determine the extent of capillary growth. Pimonidazole hydrochloride was used to assess retinal ischemia. Western blot and immunohistochemical analyses were used to assess protein expression levels and immunolocalization. Lipid peroxidation and superoxide radical formation were determined to assess oxidative changes. RESULTS. Fluvastatin treatment significantly reduced the area of the capillary-free zone (P Ͻ 0.01), decreased the formation of neovascular tufts (P Ͻ 0.01), and ameliorated retinal ischemia. These morphologic and functional changes were associated with statin effects in preventing the upregulation of VEGF, HIF-1␣, phosphorylated STAT3, and vascular expression of the inflammatory mediator ICAM-1 (P Ͻ 0.01). Superoxide production and lipid peroxidation in the ischemic retina were also reduced by statin treatment (P Ͻ 0.01). CONCLUSIONS. These data suggest the beneficial effects of statin treatment in preventing retinal neovascularization. These beneficial effects appear to result from the anti-oxidant and antiinflammatory properties of statins. (Invest Ophthalmol Vis Sci