Factors involved in the sensitivity of Stentor to colchicine, lumicolchicine, and vinblastine sulfate.

Oral regeneration by the ciliate Stentor coeruleus is inhibited by colchicine (Cc), but only at a relatively high concentration (0.9 mM); moreover, regeneration is inhibited by an even lower concentration of lumicolchicine (LCc) (0.2 mM). Together these results suggest that Cc may not be acting via tubulin binding. To evaluate this possibility we: (1) tested the effect of both drugs, and vinblastine sulfate (Vb) for comparison, on a population of labile cytoplasmic microtubules; and (2) measured the kinetics of association of all three drugs with regenerating cells. We found that Cc and Vb reduced the number of microtubules only at concentrations that blocked regeneration, whereas LCc blocked regeneration without reducing microtubule number. In addition, LCc associated with the cells much more readily than Cc, such that the cell-associated concentration of Cc that blocked regeneration was actually several fold lower than the effective concentration of LCc. We propose that common effects of Cc and LCc unrelated to tubulin binding play no more than a minor role in Cc effects on regeneration and conclude that Cc acts primarily if not exclusively via its antimicrotubule activity.

Sensitization of bleached Stentor to far UV.

Stentors are more sensitive to far UV-induced delay of oral regeneration following bleaching of their UV-absorbant cortical pigment granules. This finding supports a subcortical location of UV-sensitive sites.

Insensitivity of ciliary outgrowth in situ by Stentor to far UV.

Outgrowth of membranellar cilia in situ by the ciliate protozoan Stentor coeruleus is not affected by either far UV or by postirradiation exposure to cycloheximide, while UV does affect the outgrowth of membranellar cilia during the more elaborate process of oral regeneration in this ciliate, and cycloheximide prolongs the UV effect. These results suggest that direct damage to outgrowing ciliary shafts cannot explain the UV sensitivity of oral regeneration.

Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes.

Glutathione oxidants such as tertiary butyl hydroperoxide were shown previously to prevent microtubule assembly and cause breakdown of preassembled cytoplasmic microtubules in human polymorphonuclear leukocytes. The objectives of the present study were to determine the temporal relationship between the attachment and ingestion of phagocytic particles and the assembly of microtubules, and simultaneously to quantify the levels of reduced glutathione and products of its oxidation as potential physiological regulators of assembly. Polymorphonuclear leukocytes from human peripheral blood were induced to phagocytize opsonized zymosan at 30 degrees C. Microtubule assembly was assessed in the electron microscope by direct counts of microtubules in thin sections through centrioles. Acid extracts were assayed for reduced glutathione (GSH) and oxidized glutathione (GSSG), by the sensitive enzymatic procedure of Tietze. Washed protein pellets were assayed for free sulfhydryl groups and for mixed protein disulfides with glutathione (protein-SSG) after borohydride splitting of the disulfide bond. Resting cells have few assembled microtubules. Phagocytosis induces a cycle of rapid assembly followed by disassembly. Assembly is initiated by particle contact and is maximal by 3 min of phagocytosis. Disassembly after 5-9 min of phagocytosis is preceded by a slow rise in GSSG and coincides with a rapid rise in protein-SSG. Protein-SSG also increases under conditions in which butyl hydroperoxide inhibits the assembly of microtubules that normally follows binding of concanavalin A to leukocyte cell surface receptors. No evidence for direct involvement of GSH in the induction of assembly was obtained. The formation of protein-SSG, however, emerges as a possible regulatory mechanism for the inhibition of microtubule assembly and induction of their disassembly.

Radiosensitivity of mammalian cells. 3. Effect of suboptimal growth temperatures on recovery from radiation-induced division delay.

We investigated the effect of suboptimal growth temperatures on recovery from radiation-induced division delay in Chinese hamster cells. It was found that no recovery occurred during the time that either log-phase or synchronized populations were incubated at 4 degrees C and that injury sustained at low dose rates was cumulative over a period of 6.2 hr at low temperature. Postirradiation conditions influencing recovery from the induced division delay period are different from those affecting survival, suggesting that biochemical damage leading to division delay may be different from that leading to cell death.