ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions

Differential Inhibitory Effects of CysLT1 Receptor Antagonists on P2Y6 Receptor-Mediated Signaling and Ion Transport in Human Bronchial Epithelia

BACKGROUND: Cysteinyl leukotriene (CysLT) is one of the proinflammatory mediators released by the bronchi during inflammation. CysLTs exert their biological effects via specific G-protein-coupled receptors. CysLT(1) receptor antagonists are available for clinical use for the treatment of asthma. Recently, crosstalk between CysLT(1) and P2Y(6) receptors has been delineated. P2Y receptors are expressed in apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Previous research suggests that CysLT(1) receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors. However, the detailed molecular mechanism underlying the inhibition remains unresolved. METHODOLOGY/PRINCIPAL FINDINGS: In this study, western blot analysis confirmed that both CysLT(1) and P2Y(6) receptors were expressed in the human bronchial epithelial cell line 16HBE14o-. All three CysLT(1) antagonists inhibited the uridine diphosphate (UDP)-evoked I(SC), but only montelukast inhibited the UDP-evoked [Ca(2+)](i) increase. In the presence of forskolin or 8-bromoadenosine 3'5' cyclic monophosphate (8-Br-cAMP), the UDP-induced I(SC) was potentiated but was reduced by pranlukast and zafirlukast but not montelukast. Pranlukast inhibited the UDP-evoked I(SC) potentiated by an Epac activator, 8-(4-Chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2'-O-Me-cAMP), while montelukast and zafirlukast had no such effect. Pranlukast inhibited the real-time increase in cAMP changes activated by 8-CPT-2'-O-Me-cAMP as monitored by fluorescence resonance energy transfer imaging. Zafirlukast inhibited the UDP-induced I(SC) potentiated by N(6)-Phenyladenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-6-Phe-cAMP; a PKA activator) and UDP-activated PKA activity. CONCLUSIONS/SIGNIFICANCE: In summary, our data strongly suggest for the first time that in human airway epithelia, the three specific CysLT(1) receptor antagonists exert differential inhibitory effects on P2Y(6) receptor-coupled Ca(2+) signaling pathways and the potentiating effect on I(SC) mediated by cAMP and Epac, leading to the modulation of ion transport activities across the epithelia

The gap between clinical gaze and systematic assessment of movement disorders after stroke

Abstract Background Movement disorders after stroke are still captured by clinical gaze and translated to ordinal scores of low resolution. There is a clear need for objective quantification, with outcome measures related to pathophysiological background. Neural and non-neural contributors to joint behavior should be separated using different measurement conditions (tasks) and standardized input signals (force, position and velocity). Methods We reviewed recent literature for the application of biomechanical and/or elektromyographical (EMG) outcome measures under various measurement conditions in clinical research. Results Since 2005, 36 articles described the use of biomechanical and/or EMG outcome measures to quantify post-stroke movement disorder. Nineteen of the articles strived to separate neural and non-neural components. Only 6 of the articles measured biomechanical and EMG outcome measures simultaneously, while applying active and passive tasks and multiple velocities. Conclusion The distinction between neural and non-neural components to separately assess paresis, stiffness and muscle overactivity is not commonplace yet, while a large gap is to be bridged to attain reproducible and comparable results. Pathophysiologically clear concepts, substantiated with a comprehensive and concise measuring protocol will help professionals to identify and treat limiting factors in movement capabilities of post-stroke patients.</p

Interpreting joint moments and powers in gait

Gait analysis is becoming an increasingly important tool to provide a quantitative description of a patient's gait deviations. It is not only used to diagnose walking disorders but also for treatment selection and evaluation. While spatiotemporal, kinematic, and EMG parameters are commonly used to describe movement and muscle activity, kinetic measures are less often evaluated, even though they give insight into the moments and powers that drive human walking. As such, kinetic parameters are able to connect abnormal movement to underlying muscle malfunction and bony malalignment. This chapter focuses on the role of joint moments and powers of the lower extremities in clinical gait analysis. After a brief introduction of normal kinetic patterns, the clinical interpretation of abnormal joint moments and powers is described. Next, typical deviations in lower limb kinetics are illustrated for several patient populations, including stroke, cerebral palsy, Duchenne muscular dystrophy, anterior cruciate ligament (ACL) injury, and osteoarthritis (OA), and for patients walking with prostheses or orthotics. This section also illustrates the clinical usefulness of specific kinetic parameters in these patient populations, including their sensitivity to treatment and ability to predict treatment outcome. The chapter illustrates that the role of kinetics within clinical gait analysis deserves more attention, and potential applications should be further pursued

Strategies developed by leading EU agrifood cooperatives in their growth models

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