Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (>5min.) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high molecular weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential

X-Ray Measured Dynamics of Tycho's Supernova Remnant

We present X-ray proper-motion measurements of the forward shock and reverse-shocked ejecta in Tycho's supernova remnant, based on three sets of archival Chandra data taken in 2000, 2003, and 2007. We find that the proper motion of the edge of the remnant (i.e., the forward shock and protruding ejecta knots) varies from 0".20 yr^{-1} (expansion index m=0.33, where R = t^m) to 0".40 yr^{-1} (m=0.65) with azimuthal angle in 2000-2007 measurements, and 0".14 yr^{-1} (m=0.26) to 0".40 yr^{-1} (m=0.65) in 2003-2007 measurements. The azimuthal variation of the proper motion and the average expansion index of ~0.5 are consistent with those derived from radio observations. We also find proper motion and expansion index of the reverse-shocked ejecta to be 0".21-0".31 yr^{-1} and 0.43-0.64, respectively. From a comparison of the measured m-value with Type Ia supernova evolutionary models, we find a pre-shock ambient density around the remnant of <~0.2 cm^{-3}

Tycho SN 1572: A Naked Ia Supernova Remnant without Associated Ambient Molecular Cloud

The historical supernova remnant (SNR) Tycho SN 1572 originates from the explosion of a normal Type Ia supernova which is believed to have originated from a carbon-oxygen white dwarf in a binary system. We analyze the 21cm continuum, HI and 12CO-line data from the Canadian Galactic Plane Survey in the direction of SN 1572 and surrounding region. We construct HI absorption spectra to SN 1572 and three nearby compact sources. We conclude that SN 1572 has no molecular cloud interaction, which argues against previous claims that a molecular cloud is interacting with the SNR. This new result does not support a recent claim that dust, newly detected by AKARI, originates from such a SNR-cloud interaction. We suggest that the SNR has a kinematic distance of 2.5 - 3.0 kpc based on a nonlinear rotational curve model. Very-high-energy Gamma-ray emission from the remnant has been detected by the VERITAS telescope, so our result shows that its origin should not be an SNR-cloud interaction. Both radio and X-ray observations support that SN 1572 is an isolated Type Ia SNR.Comment: 14 pages, 4 figs, accepted by ApJ Letter

The Cholinergic Pathway and MitoKATP Induce UCP4 Expression Involved in Neuroprotection of FN Stimulation in Rats

Background Electrical stimulation of the cerebellar fastigial nucleus (FN) reduces the infarct size induced by middle cerebral artery occlusion in rats. FN stimulation confers long‐lasting protection from brain injury; however, its underlying mechanism is not yet understood. We aimed to elucidate the mechanism by which FN stimulation exerts neuroprotection. We hypothesized that the neuroprotective effect of FN stimulation involves activation of cholinergic pathways, which increases reactive oxygen species (ROS) production by opening mitochondrial K+ATP channels, thus leading to an increase in UCP4 (uncoupling protein 4) expression and subsequent neuroprotection. Methods FN stimulation was performed for 1 hour in rats. The UCP4 protein and mRNA levels were measured by western blot, dot blot, and in situ hybridization. Carbachol was applied following UCP4‐promoter tdTomato reporter vector transfection of the rat primary cortical cell culture (in vitro) and rat brain (in vivo). We observed cellular UCP4 expression using fluorescence microscopy. UCP4 expression in the cell culture in response to diazoxide application was determined by a reverse transcription‐polymerase chain reaction and western blotting. Results Whereas FN stimulation increased UCP4 protein and mRNA levels, carbachol administration induced UCP4 expression in vitro and in vivo. The attenuation of this effect by atropine suggests that FN‐induced UCP4 expression involves the cholinergic pathway. The opening of mitochondrial K+ATP channels with diazoxide increased the production of ROS and led to increased UCP4 expression. In contrast, quenching ROS with superoxide dismutase reversed the effect of diazoxide on UCP4 expression. Therefore, the opening of mitochondrial K+ATP channels increased ROS production, which subsequently enhanced UCP4 expression and attenuated ROS generation. Conclusion Neuroprotective effect of FN stimulation involves activation of the cholinergic pathways, which increases ROS production by opening mitochondrial K+ATP channels, leading to increased expression of neuroprotective UCP4