Laboratory-size x-ray microscope using Wolter mirror optics and an electron-impact x-ray source.

We developed a laboratory-size three-dimensional water-window x-ray microscope using condenser and objective grazing incidence Wolter type I mirrors, an electron-impact-type x-ray source, and a back-illuminated CCD. The imaging system was improved for practical applications in life science research fields. Using a new objective mirror with reduced figure errors, a resolution limit of 3.1 line pairs/µm was achieved for two-dimensional transmission images and sub-micrometer-scale three-dimensional structures were resolved. Incorporating a cryogenic stage into the x-ray microscope, we observed biological samples embedded in ice to evaluate the usefulness of observation in the water-window region and multi-energy observation was demonstrated using an x-ray source with multiple x-ray tubes.

Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12.

OBJECTIVE: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells. METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques. RESULTS: Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis. CONCLUSIONS: Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.

Laboratory-size three-dimensional x-ray microscope with Wolter type I mirror optics and an electron-impact water window x-ray source.

We constructed a laboratory-size three-dimensional water window x-ray microscope that combines wide-field transmission x-ray microscopy with tomographic reconstruction techniques, and observed bio-medical samples to evaluate its applicability to life science research fields. It consists of a condenser and an objective grazing incidence Wolter type I mirror, an electron-impact type oxygen Kα x-ray source, and a back-illuminated CCD for x-ray imaging. A spatial resolution limit of around 1.0 line pairs per micrometer was obtained for two-dimensional transmission images, and 1-µm scale three-dimensional fine structures were resolved.

Akt and Ca2+ signaling in endothelial cells.

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.

Usefulness of stress myocardial perfusion imaging for evaluating asymptomatic patients after coronary stent implantation.

BACKGROUND: Stent implantation in coronary angioplasty has reduced the rate of restenosis, but many patients still undergo follow-up coronary angiography (CAG). The present study was a multi-center retrospective analysis of the usefulness of stress single photon emission computed tomography (SPECT) compared with follow-up CAG in stent-implanted patients who remained asymptomatic during the follow-up period. METHODS AND RESULTS: The study group of 103 patients underwent both SPECT and CAG at 4-9 months after stent implantation. Restenosis occurred in 20 (19%) of 106 vessel territories, and a reversible perfusion defect was found in 32 (30%) territories. Sensitivity, specificity, positive and negative predictive values, and accuracy of SPECT were 65%, 78%, 41%, 91%, and 76%, respectively. The accuracy was lower in territories with a prior myocardial infarction (71%), in the left circumflex artery (58%), and in cases with three-vessel disease (63%). The negative predictive value was high, but 7 false negative cases included 4 cases with prior myocardial infarction, and 2 cases with reversible defects in other vessel territories. CONCLUSIONS: Stress SPECT imaging is a useful tool for following up patients with coronary stent implantation, and follow-up CAG could be omitted in patients with negative SPECT imaging, no prior myocardial infarction, one- or two-vessel disease, and sufficient stress loading.