Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20–45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO3–, NO2–, HNO, ONOO–, NO2, OCl–, and H2O2. The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.National Science Foundation (U.S.) (CHE-0611944)National Institutes of Health (U.S.) (K99GM092970

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology

Purification and Structural Study of the Voltage-Sensor Domain of the Human KCNQ1 Potassium Ion Channel

KCNQ1 (also known as K_V7.1 or K_VLQT1) is a voltage-gated potassium channel modulated by members of the KCNE protein family. Among multiple functions, KCNQ1 plays a critical role in the cardiac action potential. This channel is also subject to inherited mutations that cause certain cardiac arrhythmias and deafness. In this study, we report the overexpression, purification, and preliminary structural characterization of the voltage-sensor domain (VSD) of human KCNQ1 (Q1-VSD). Q1-VSD was expressed in <i>Escherichia coli</i> and purified into lyso-palmitoylphosphatidylglycerol micelles, conditions under which this tetraspan membrane protein yields excellent nuclear magnetic resonance (NMR) spectra. NMR studies reveal that Q1-VSD shares a common overall topology with other channel VSDs, with an S0 helix followed by transmembrane helices S1–S4. The exact sequential locations of the helical spans do, however, show significant variations from those of the homologous segments of previously characterized VSDs. The S4 segment of Q1-VSD was seen to be α-helical (with no 3₁₀ component) and underwent rapid backbone amide H–D exchange over most of its length. These results lay the foundation for more advanced structural studies and can be used to generate testable hypotheses for future structure–function experiments

White Paper: functionality and efficacy of wrist protectors in snowboarding—towards a harmonized international standard

The wrist is the most frequently injured body region among snowboarders. Studies have shown that the risk of sustaining a wrist injury can be reduced by wearing wrist protection. Currently, there are a wide variety of wrist protection products for snowboarding on the market that offer a range of protective features. However, there are no minimum performance standards for snowboarding wrist protectors worldwide. The International Society for Skiing Safety convened a task force to develop a White Paper to evaluate the importance and necessity of a minimum performance for all wrist protectors used in snowboarding. The White Paper outlines the need for a general framework for a harmonized international standard and reviews the existing evidence. Therefore, this White Paper may serve as a common base for future discussions. The broader goal of developing and implementing such a standard is to reduce the incidence and the severity of wrist injuries in snowboarding without increasing the risk of adverse events, such as upper arm or shoulder injury. The European standard for inline skating wrist protectors (EN 14120) can serve as a starting point for efforts related to a standard for snowboard wrist protectors, but certain modifications to the standard would be required. It is hypothesized that implementation of a snowboarding wrist protector standard would result in fewer and less severe wrist injuries in the sport and could translate into more riding days for healthy snowboarders and significant health-care costs savings