Synergistic antitumor activity from two-stage delivery of targeted toxins and endosome-disrupting nanoparticles.

Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of gelonin to be focused on tumors with significantly reduced potential for off-target toxicity.

'Injecting' yeast.

Yeast is a powerful genetic model system, but its rigid cell wall has prohibited microinjection. Using microfabricated channels to constrain the fission yeast Schizosaccharomyces pombe, we sheared local regions of individual cells with a piezoelectric unit. The cells remained viable, we detected actin patches in the cell after introduction of fluorescent phalloidin into the medium, and the cytokinetic ring was disrupted after injection of the myosin II inhibitor blebbistatin.

Integrin-driven actin polymerization consolidates long-term potentiation.

Long-term potentiation (LTP), like memory, becomes progressively more resistant to disruption with time after its formation. Here we show that threshold conditions for inducing LTP cause a rapid, long-lasting increase in polymerized filamentous actin in dendritic spines of adult hippocampus. Two independent manipulations that reverse LTP disrupted this effect when applied shortly after induction but not 30 min later. Function-blocking antibodies to beta1 family integrins selectively eliminated both actin polymerization and stabilization of LTP. We propose that the initial stages of consolidation involve integrin-driven events common to cells engaged in activities that require rapid morphological changes.

Structural and functional alterations of hepatocytes during transient phalloidin-induced cholestasis in the rat.

To study the relationship between the dynamic actin web and bile secretion, we developed an acute model of cholestasis, using phalloidin, and examined sequential morphologic and biochemical events in rat liver. Biliary function (bite flow, bile, and canalicular membrane components) and cellular integrity (release of hepatic enzymes in serum and bile, canalicular structure, and microfilaments distribution) in rats given a single iv dose of phalloidin (0.8 mg/kg body weight) were assessed at 15, 45, and 90 min, 24 hr, and 5 days postinjection. Bile flow decreased significantly at 45 and 90 min, but cholestasis was transient since bile secretion returned to control levels at 24 hr. The biliary bile acid secretion rate was not modified during the same time period, indicating that cholestasis may have been due to impairment of the bile acid independent component of bile flow. Serum alanine aminotransferase and lactate dehydrogenase as well as biliary alkaline phosphatase and alkaline phosphodiesterase-1 activities were not altered by phalloidin treatment. These data, coupled with morphologic studies, provide no evidence of cell damage. Electron microscopy revealed that the pericanalicular actin web in both centrilobular and periportal hepatocytes was increased at 90 min and further enlarged at 24 hr and 5 days after phalloidin injection. At all time periods, the canalicular structure was well preserved. Na+K+ -ATPase and Mg2+ -ATPase activities in membrane fractions enriched in bile canalicular complexes decreased significantly at 15 min and remained low up to Day 5. Mg2+ -ATPase activity returned to control levels by Day 5. The lipid constituents of liver cell membranes enriched in canalicular complexes showed no significant variations 90 min after toxin treatment but, at 24 hr, phospholipid content rose and membrane fluidity increased. These results clearly indicate that the bile flow variation after a single low dose of phalloidin can be dissociated from specific pericanalicular microfilament distribution, lending further support to the view that normal biliary function is not strictly dependent on the integrity of the actin filament network.

Modulation of bronchial epithelial cell barrier function by in vitro ozone exposure.

The epithelial cells lining the small, peripheral airways function as important targets for the action of inspired ozone. Loss of epithelial barrier integrity in these regions is a common element in ozone-induced airway inflammation. To investigate the direct effect of ozone on epithelial barrier function, canine bronchial epithelial (CBE) cells grown with an air interface were exposed for 3 hr to 0.2, 0.5, or 0.8 ppm ozone or to air. Mannitol flux, used as an index of paracellular permeability, increased above air controls by 461%, 774%, and 1172% at the three ozone concentrations, respectively. Transcellular electrical resistance exhibited a dose-related decrease. The immediate effect of 0.8 ppm ozone on permeability was significantly inhibited by preincubation for 48 hr in the presence of 1 ng/ml vitamin E (33%) or 1 microM vitamin A (34%). Responses to 0.5 ppm or 0.8 ppm were inhibited by pretreatment of the cells with 0.1 microM of the actin polymerizing agent phalloidin (34% and 25% inhibition, respectively). The increases in permeability induced by 0.2 and 0.5 ppm ozone were attenuated by 54% and 22%, respectively, at 18 hr after exposure, whereas that to 0.8 ppm was further enhanced by 42% at this time. The effects of ozone are modulated by the availability of antioxidants to the cells and appear to be associated with cytoskeletal dysfunction in CBE cells. The data are consistent with a loss of barrier function linked to a direct oxidative effect of ozone on individual CBE cells and indicate that the reversible or progressive nature of this effect is dose dependent.

Effect of ethacrynic acid on aqueous outflow dynamics in monkeys.

PURPOSE: To determine the long-term effect of ethacrynic acid (ECA) on aqueous outflow dynamics in ocular normotensive monkeys. METHODS: (1) Twelve cynomolgus monkeys received 10 microliters of 2.5 mM (= 7.5 micrograms) ECA intracamerally in one eye, vehicle in the other; outflow facility (perfusion) was determined at 1 hour, 24 hours, or 1 week, and intraocular pressure (IOP; applanation) at 24 hours, 1 week, and 6 to 7 weeks later. Six other cynomolgi received 10 microliters of 0.13 or 1.3 mM phalloidin in one eye 45 minutes before receiving ECA OU; facility was measured 1 hour after ECA. (2) Groups of five rhesus monkeys underwent intracameral injection of 2.5, 5.0, or 10 micrograms ECA in one eye, vehicle in the other, with IOP measured hours to weeks thereafter. (3) Five rhesus monkeys received 540 micrograms of ECA unilaterally as a 30-microliter topical drop once daily for 4 days. On the first and fourth treatment days, baseline IOP, pupil diameter, and refraction were measured immediately before and again at 2, 4, and 7 hours after topical treatment. RESULTS: (1) ECA divided by vehicle facility averaged 1.83 +/- 0.23 (SEM) (P < 0.02, n = 6), 1.50 +/- 0.27 (P < 0.11, n = 7), and 1.05 +/- 0.10 (n = 7) at 1 hour, 24 hours, and 1 week, respectively. IOP was 1 to 2 mm Hg lower in ECA eyes 24 hours (P < 0.05, n = 5) and 6 to 7 weeks (P < 0.05, n = 7) after treatment. Phalloidin did not diminish the 1-hour ECA facility effect. (2) Five (but not 2.5 or 10) micrograms of ECA lowered IOP 1 to 3 mm Hg, starting at 2 hours and lasting up to 48 hours. The maximum ECA effect (-2.6 +/- 0.25 mm Hg; P < 0.001, n = 5) occurred at 4 hours. IOP, corneal thickness and endothelial cell count, and anterior chamber depth were not significantly different 8 weeks after 5 micrograms ECA or vehicle. (3) Once-daily unilateral topical application of 540 micrograms of ECA induced no change in IOP, refraction, or pupil diameter compared to contralateral vehicle-treated control eyes. There were no significant ECA-related ocular bio-microscopic abnormalities. CONCLUSIONS: ECA may lower IOP and increase outflow facility longer than previously thought, but not by affecting meshwork actin filaments.

Phalloidin-induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation.

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.

Decreased sensitivity to phalloidin of normal-looking rat hepatocytes after short-term 2-acetylaminofluorene feeding.

Male F344/DuCrj rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF) for 1 or 3 weeks, and then fed a basal diet for 2 days, 2 weeks, 8 weeks, 22 weeks or 36 weeks. Hepatocytes were isolated from the liver by collagenase perfusion, and their sensitivity to phalloidin, in terms of the formation of multiple cytoplasmic blebs, was examined. The sensitivity of gamma-glutamyltransferase (GGT)-negative hepatocytes decreased on the 22nd and 36th weeks after withdrawal of 2-AAF feeding, and that of GGT-positive cells decreased on the 36th week. Induction of a small number of foci positive for the placental form of glutathione S-transferase (GSTP) was observed in the liver of all rats on the 8th, 22nd and 36th weeks after the withdrawal of the carcinogen. However, the total area of the foci was estimated to account for less than 0.2% of liver tissues even on the 36th week. Therefore, the decrease in phalloidin sensitivity of hepatocytes, particularly of GGT-negative hepatocytes, on the 22nd and 36th weeks after 2-AAF withdrawal is suggested to be a result of a decrease in the sensitivity of otherwise normal-looking hepatocytes, which may be precursors of the cells forming the preneoplastic foci.