Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium. MATERIALS AND METHODS Reagents Unless otherwise specified, reagents (including S1P and thrombin) were obtained from Sigma (St. Louis, MO). Mouse anticortactin antibody (4F11) was obtained from Upstate Biotechnology (Lake Placid, NY).

Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0 nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wild-type SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a “toxic channel” mechanism presents a new therapeutic direction for ALS research.

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We have been investigating the use of the alpha-emitting radionuclide 2,2 Bi against microscopic carcinoma. Our in vitro studies show that 212 Bi is 2 to 4 times more effective in eradicating microscopic cells grown in monolayer or multicellular spheroid. Autoradiographs show that 212 Bi diffuses within the spheroids by 2 hours after exposure. There was no difference in cell kill if cells were grown in monolayer or 100 μπι and 800 μιη spheroids. From our study, 212 Bi appears to be a suitable candidate to investigate for clinical use against microscopic carcinoma.