Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

Energy distributions of particles striking the cathode in a glow discharge

Charge-exchange collision in the cathode fall region of an abnormal glow discharge is assumed to be the mechanism which limits the energy of the ions and generates the energetic neutrals bombarding the cathode. The model used by W. D. Davis and T. A. Vanderslice Phys. Rev. 131 219 (1963) for the calculation of the ion distribution was not applied to calculate the distribution of neutrals. A transport formulation is proposed that allows the calculation of both the distributions of energetic ions and fast neutrals.This work is part of a research project under the auspices of the Spanish-CAICYT (Proyecto No. 599), in collaboration with the University of Salford (financially supported by The British Council and the Spanish MEC)