cytochalasin-d and thiazolyl-blue

Exogenous thymosin beta-4 (Tbeta(4)) has been shown to inhibit the apoptosis in nontransformed human corneal epithelial cells that is triggered by ethanol. The purpose of this study is to examine whether exogenous Tbeta(4) protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H(2)O(2)) and to elucidate its mechanism of action.. HCE-T cells were incubated without or with the recombinant histidine-tagged Tbeta(4) produced by Escherichia coli before the addition of FasL or H(2)O(2). Cell viability was determined by MTT or MTS assay, and activation of caspase-8, -9, and -3 was examined by colorimetric and fluorescent substrate cleavage assays. The internalization of exogenous Tbeta(4) in HCE-T cells was analyzed by immunofluorescence staining. Cytochalasin D, an actin depolymerization agent, was added to examine whether the actin cytoskeleton is involved in Tbeta(4) entry and whether the internalization of this peptide is crucial for its cytoprotection.. The death of HCE-T cells induced by both FasL and H(2)O(2) was dramatically reduced by the recombinant Tbeta(4) pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H(2)O(2)-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tbeta(4). Of note, internalization of this G-actin-sequestering peptide into HCE-T cells was found to be essential in cell death prevention, in that disruption of the cellular entry of Tbeta(4) by cytochalasin D abrogated its cytoprotective effects.. This is the first report to demonstrate that the internalization of exogenous Tbeta(4) is essential for its antiapoptotic activity in human corneal epithelial cells.

Topics: Animals; Apoptosis; Blotting, Western; Caspases; Cell Culture Techniques; Cell Line, Transformed; Cytochalasin D; Endocytosis; Epithelium, Corneal; Fas Ligand Protein; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Hydrogen Peroxide; Maximum Tolerated Dose; Rabbits; Recombinant Proteins; Simian virus 40; Tetrazolium Salts; Thiazoles; Thymosin

The structure of human fibroblasts have been characterised in vitro by atomic force microscopy (AFM) operated in the imaging or in the force versus distance (F-d) modes. The choice of cell substrate is important to ensure good adhesion. Of greater significance in the context of AFM analysis, is the observation that the substrate affects the imaging conditions for in vitro analysis of live cells. For instance, very rarely will glass coverslips lead to acceptable outcomes (i.e., resolved cytoskeletal structure). Activated tissue culture dishes, on the other hand, promote conditions that routinely result in good quality images. Those conditions are then unaffected by adoption of relatively high force loadings (more than 10 nN), large fields of view (100 x 100 microm2) and high scan speeds (up to ca. 200 microm/sec), all of which exceed values recommended in the literature. Plasma membranes are fragile in the context of AFM analysis (F-d analysis gives an equivalent Young's Modulus of ca. 5 kPa). However, the present work suggests that fragility per se need not be a problem, rather it is the adhesive interactions with the tip, which under some circumstances may exceed 20 nN, that are the source of poor imaging conditions. The present results, being supported by a qualitative model, suggest that the activated substrate acts as a preferential scavenger of cellular debris thus preventing the tip from biofouling, and will therefore promote low adhesion between tip and membrane. Good imaging conditions provide non-destructive in vitro information about cytoskeletal structure and dynamics, as shown in two examples concerned with cytochalasin treatment and with the MTT assay.

Topics: Cell Division; Cells, Cultured; Coloring Agents; Cytochalasin D; Fibroblasts; Humans; Microscopy, Atomic Force; Tetrazolium Salts; Thiazoles