ABSTRACT:We have developed a near-IR reversible fluorescent probe containing an organoselenium functional group that can be used for the highly sensitive and selective monitoring of peroxynitrite oxidation and reduction events under physiological conditions. The probe effectively avoids the influence of autofluorescence in biological systems and gave positive results when tested in both aqueous solution and living cells. Real-time images of cellular peroxynitrite were successfully acquired. P eroxynitrite (ONOO À ) performs as a strong oxidizing agent in physiological and pathological processes. 1À3 In vivo, abnormally high concentrations of ONOO À are formed from the fast reaction between nitric oxide (NO) and superoxide anion (O 2 À ), which requires no enzymatic catalysis. 4 The peroxynitrite anion is relatively stable, but the acid form (ONOOH) rapidly decays to nitrate. Although the half-life of ONOOH is ∼1 s at pH 7.40, 5 the oxidative species contributes to signal transduction, homeostasis regulation, and oxidative damage, which forms a unique biological oxidationÀreduction cycle indicating human health and disease.6 Therefore, the development of reversible detection technology for peroxynitrite would have important biomedical significance. In comparison with other technologies, fluorescence microscopy provides greater sensitivity, less invasiveness, and more convenience.7 Especially the use of near-IR (NIR) light (650À900 nm) allows deep penetration into tissues and efficaciously avoids the influence of bioautofluorescence. However, there exists a major obstacle to the design of novel fluorescent probes for ONOO À , namely, the nitro group, which is considered to be a strong quencher for fluorophores.3 To date, only a few fluorescent probes for ONOO À detection have been reported. 8 Thus, we anticipate widespread interest in a redoxreversible NIR fluorescent probe, which would exhibit much more value for visualizing cycles of redox signaling and stress caused by peroxynitrite.9 Here we report a redox-responsive NIR fluorescent probe for continuous monitoring of ONOO À . ONOO À is modulated by cellular antioxidant defense systems, 1,10 in which selenium (Se) plays an important role as the active site of the antioxidant enzyme glutathione peroxidase (GPx).11 GPx can catalyze the reduction of ONOO À by glutathione (GSH) via a unique ping-pong mechanism.12 Taking the advantage of this, we mimicked the catalytic cycle and developed an NIR fluorescent probe containing an organoselenium moiety that can be used for reversible peroxynitrite detection.As an overall strategy, cyanine (Cy), an NIR fluorescent dye with a high extinction coefficient, 13 was selected as a signal transducer, while 4-(phenylselenyl)aniline (PSe) was selected as a modulator because it can respond sensitively to ONOO À . 11,14Following the ping-pong mechanism, 12 we designed and synthesized a new NIR reversible fluorescent probe (Cy-PSe) for detection of ONOO À in living cells through a fast photoinduced electron transfer (PET) process. ...

Mitochondrial dysfunction is an early pathological feature of Alzheimer’s disease (AD). The underlying mechanisms and strategies to repair it remain unclear. Here, we demonstrate for the first time the direct consequences and potential mechanisms of mitochondrial functional defects associated with abnormal mitochondrial dynamics in AD. Using cytoplasmic hybrid (cybrid) neurons with incorporated platelet mitochondria from AD and age-matched non-AD human subjects into mitochondrial DNA (mtDNA)-depleted neuronal cells, we observed that AD cybrid cells had significant changes in morphology and function; such changes associate with altered expression and distribution of dynamin-like protein (DLP1) and mitofusin 2 (Mfn2). Treatment with antioxidant protects against AD mitochondria-induced extracellular signal-regulated kinase (ERK) activation and mitochondrial fission-fusion imbalances. Notably, inhibition of ERK activation not only attenuates aberrant mitochondrial morphology and function but also restores the mitochondrial fission and fusion balance. These effects suggest a role of oxidative stress-mediated ERK signal transduction in modulation of mitochondrial fission and fusion events. Further, blockade of the mitochondrial fission protein DLP1 by a genetic manipulation with a dominant negative DLP1 (DLP1K38A), its expression with siRNA-DLP1, or inhibition of mitochondrial division with mdivi-1 attenuates mitochondrial functional defects observed in AD cybrid cells. Our results provide new insights into mitochondrial dysfunction resulting from changes in the ERK-fission/fusion (DLP1) machinery and signaling pathway. The protective effect of mdivi-1 and inhibition of ERK signaling on maintenance of normal mitochondrial structure and function holds promise as a potential novel therapeutic strategy for AD.

he directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology-the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 10(15) transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200800 transformants in one library. The triple code utilizes structural (X-ray) and consensus-derived sequence data, and is therefore designed to match the steric and electrostatic characteristics of the particular enzyme. Using this approach, limonene epoxide hydrolase has been successfully engineered as stereoselective catalysts in the hydrolytic desymmetrization of meso-type epoxides with formation of either (R,R)- or (S,S)-configurated diols on an optional basis and kinetic resolution of chiral substrates. Crystal structures and docking computations support the source of notably enhanced and inverted enantioselectivity