Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound.

An increase in cytoplasmic calcium (Ca(2+) increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca(2+) in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca(2+) in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca(2+) was observed; however, no increase in Ca(2+) was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca(2+) in cells subjected to pulsed ultrasound.

Thermally sensitive cationic polymer nanocapsules for specific cytosolic delivery and efficient gene silencing of siRNA: swelling induced physical disruption of endosome by cold shock.

Pluronic/poly(ethylenimine) (PEI2K) nanocapsules (NCs) exhibiting a thermally reversible swelling/deswelling volume expansion behavior were synthesized and used for an siRNA delivery nanocarrier as well as an effective endosome breaking agent. Pluronic/PEI2K nanocapsules were in a collapsed state with an average size of 118.9+/-15.3 nm at 37 degrees C, but in a swollen state with that of 412.3+/-83.2 nm at 15 degrees C. The collapsed Pluronic/PEI2K NCs having a highly positive surface zeta potential value were utilized to load siRNA-PEG conjugate on the surface via electrostatic interactions. The siRNA-PEG/NCs nanocomplexes were transfected to the cells at 37 degrees C for efficient cellular uptake. The transfected cells were treated with a brief cold shock to induce an abrupt volume expansion of the NCs within an endosome compartment to physically burst out the endosomal membrane. Far efficient gene silencing effects for both green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) were observed after the cold shock treatment to the transfected cells.

HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses.

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.

The cAMP-binding proteins of Leishmania are not the regulatory subunits of cAMP-dependent protein kinase.

The most commonly used method to determine the cAMP binding activity in cytosolic extracts of promastigotes of Leishmania spp. underestimated by approximately 11.5-fold the total amount of [(3)H]cAMP bound, when compared with results obtained by the modified Millipore filter technique. Three cAMP-binding proteins (BPI, BPII and BPIII) were partially purified and characterized. The native molecular masses of BPI, BPII and BPIII were estimated to be 105, 155 and 145 kDa, respectively. The binding of [(3)H]cAMP to these proteins was affected to different extents by several cAMP analogues. Antibodies directed against the types I and II regulatory subunits of PKA did not cross-react with the leishmanial extract. Photoaffinity labeling of the cytosolic extracts with 8-N(3)-[(32)P]cAMP specifically labeled a band of M(r) 116000 and a band of M(r) 80000 partially saturable by cAMP. From these results, it is concluded that the leishmanial cAMP-binding proteins appear to belong to a different class distinct from the regulatory subunits of cAMP-dependent protein kinases.

Differential mechanism of retention of Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) by brain and tumor: a novel radiopharmaceutical for positron emission tomography imaging.

The reductive retention of 62Cu-PTSM was comparatively studied in the brain and Ehrlich ascites tumor cells by electron spin resonance spectrometry and nonradioactive Cu-PTSM. In the brain, only the mitochondrial fraction showed the ability to reduce Cu-PTSM, and the other subcellular fractions did not. In contrast, the cytosolic fraction of Ehrlich ascites tumor cells was the specific site of Cu-PTSM reduction. It was therefore considered that the retention of Cu-PTSM in the brain is closely related to mitochondrial reduction, most probably involving the mitochondrial electron transport system.