Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer.

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser-ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal-regulated kinase was activated at the core of contracting platelet aggregates. SUMMARY: Background The dynamic features of thrombus formation have been visualized by conventional video widefield microscopy or confocal microscopy in live mice. However, owing to technical limitations, the precise spatiotemporal regulation of intracellular signaling molecule activities, which have been extensively studied in vitro, remains elusive in vivo. Objectives To visualize, by the use of two-photon excitation microscopy of transgenic mice expressing Förster resonance energy transfer (FRET) biosensors for extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), ERK and PKA activities during thrombus formation in laser-injured subcutaneous arterioles. Results When a core of densely packed platelets had developed, ERK activity was increased from the basal region close to the injured arterioles. PKA was activated at the downstream side of an unstable shell overlaying the core of platelets. Intravenous administration of a MEK inhibitor, PD0325901, suppressed platelet tethering and dislodged platelet aggregates, indicating that ERK activity is indispensable for both initiation and maintenance of the thrombus. A cAMP analog, dbcAMP, inhibited platelet tethering but failed to dislodge the preformed platelet aggregates, suggesting that PKA can antagonize thrombus formation only in the early phase. Conclusion In vivo imaging of transgenic mice expressing FRET biosensors will open a new opportunity to visualize the spatiotemporal changes in signaling molecule activities not only during thrombus formation but also in other hematologic disorders.

Development of an automated ballast water treatment verification system utilizing fluorescein diacetate hydrolysis as a measure of treatment efficacy.

Methods for verifying ballast water treatments in foreign vessels are needed to protect the Great Lakes from the discharge of live non-native organisms or pathogens. A prototype automated viability test system using fluorescein diacetate (FDA), a membrane permeable fluorogen, to differentiate live from dead bacteria and algae is described. The automated fluorescence intensity detection device (AFIDD) captures cultured algae or organisms in Detroit River water (simulated ballast water) on 0.2 µm filters, backwashes them from the filter into a cuvette with buffer and FDA for subsequent fluorescence intensity measurements, and washes the filters with sterile water for serial automated reuse. Preliminary manual versions of these procedures were also tested. Tests of various buffers determined N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine (BES) and 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0 to be the best buffers, causing the least spontaneous FDA breakdown without inhibiting enzymatic activity. Fluorescence in the presence of live organisms increased linearly over time, and the rate of increase was dependent on the sample concentration. Following simulated ballast water treatments with heat or chlorine, the fluorescence produced by Detroit River samples decreased to near control (sterile water) levels. Automated measurements of FDA hydrolysis with a reusable filter backwash system should be applicable to near real-time remote-controlled monitoring of live organisms in ballast water.

Depletion of dietary n-3 fatty acid affects the level of cyclic AMP in rat hippocampus.

Prolonged depletion of dietary n-3 fatty acid induces a neurological disturbance. To ascertain the deficit of neurotransmission at the time of n-3 deficiency, the concentrations of cAMP and inositol triphosphate, and the activities of protein kinases A and C were examined in vitro in rat hippocampus. Furthermore, the saturation binding study of [3H]quinuclidinyl benzilate, a specific antagonist to muscarinic cholinergic receptor, was performed. Rats were fed a safflower oil diet as the deficient group and a soybean oil diet as the control group. Hippocampi were obtained from rats in the 3rd generation in the deficient group and in the 2nd generation in the control group. Dietary effect was not observed in the parameters except for the concentration of cAMP, which was significantly higher in the deficient group than in the control group.