The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm

Single-walled carbon nanotube interactions with HeLa cells

This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports

Retrograde transport of protein toxins under conditions of COPI dysfunction

AbstractRetrograde transport dependent on coat protein I (COPI) was impaired using two different approaches and the effects on the retrograde transport of protein toxins were investigated. One approach was to study ldlF cells that express a temperature-sensitive defect in the ϵ-COP subunit of COPI. The second approach was to treat cells with 1,3-cyclohexanebis(methylamine) (CBM), a drug that interferes with the binding of COPI to Golgi membranes. With both approaches, cells remained sensitive to a variety of protein toxins regardless of whether the toxins contained a KDEL motif. Moreover, cholera toxin, which contains a KDEL sequence, was observed by immunofluorescence microscopy to enter the endoplasmic reticulum of Vero cells in the presence of CBM. These data support published evidence indicating the presence in cells of a COPI- and KDEL receptor-independent pathway of retrograde transport from the Golgi complex to the endoplasmic reticulum. In addition, the results suggest that certain toxins containing a KDEL motif may use either the COPI-dependent or COPI-independent pathway of retrograde transport