Effects of excess thyroid hormone on cell death, cell proliferation, and new neuron incorporation in the adult zebra finch telencephalon.

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T4) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long-projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [3H]-thymidine labeling, co-labeled with anti-TR antibody. Acute T4 treatment dramatically increased the number of pyknotic and TUNEL-positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub-telencephalic regions, suggesting that excess T4 augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T4 treatment at a dosage that stimulated cell death, although [3H]-labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T4 treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T4 affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior.

Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.

The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory. Twenty sites were sampled; defensin was found in 100% of the sites and ranged in amount from 270-2000 ng/site. The large local concentrations of defensins, estimated in the mg/ml range, may have major effects on the microbiology of the gingival crevice.

Aggregation-dependent changes in susceptibility of Dictyostelium discoideum to amphotericin B.

In this study, the polyene antibiotic amphotericin B was used to induce changes in permeability and viability of the eucaryotic microorganism Dictyostelium discoideum. The results show that as the cells progress from the growth phase through stationary phase to eventual aggregation, they become increasingly resistant to both permeability changes and lysis. Plasma membranes were prepared from cells harvested at exponential growth, stationary phase, and the aggregation-competent stage, and both the neutral lipid and phospholipid content were determined. An increase in the neutral lipid and a decrease in the phospholipid were observed as the cells progressed from growth into aggregation, with the overall result that the phospholipid-sterol ratio decreased during this period. The extent of amphotericin B binding by cells from the different stages was also determined. Aggregation cells exhibited a small but significant increase in binding compared to cells from either of the other two stages. The permeability changes produced by the drug were measured as a function of temperature. Exponentially growing cells showed a marked temperature dependence for the drug effect, whereas aggregating cells did not. Rates of inactivation by the drug were also determined over a range of temperatures. With exponentially growing cells, the rate of inactivation was temperature dependent, whereas with aggregating cells it was not. Finally, drug binding for both growing and aggregating cells was temperature dependent. Thus, no binding was observed for cells of either type at 4 degrees C. This finding suggests that the differences in the temperature dependence of the permeability changes and inactivation between the cells involve parameters other than drug binding.

Time-dependent changes in Dictyostelium discoideum adenylate cyclase activity upon incubation with ATP.

In this study we report that preincubation of Dictyostelium discoideum membrane-bound adenylate cyclase with ATP over the concentration range 0.5 to 100 mM results in a loss of catalytic activity and that this effect persists even after removal of ATP. An analysis of the time course of this effect shows that, at 25 mM ATP, a 5- to 10-min preincubation results in 50% loss of activity. Additional studies on this effect showed that anhydride bond cleavage of ATP occurs during the preincubation. However, loss of catalytic activity is not porduced by ADP, AMP, cAMP, adenosine, pyrophosphate, or phosphate either separately or in pairs. Further, using the structural analogs adenosine 5'-(alpha, beta-methylene)triphosphate and adenyl-5'-yl imidodiphosphonate, we show that there is a direct correlation between alpha-beta-phosphoanhydride bond cleavage and the loss of catalytic activity. These results can be interpreted in terms of two classes of reaction mechanisms: either those involving covalent modifications or those involving a ligand-induced slow conversion of the adenylate cyclase from an active to an inactive form. Additional studies show that the addition of AMP to the reaction mixture, as well as removal of the membrane-bound 5'-nucleotidase activity, can prevent the loss of cyclase activity. These results suggest not only that adenylate cyclase activity is related to the AMP:ATP ratio but that the cyclase activity can be modified by the level of 5'-nucleotidase activity. Studies on the duration of the loss of activity produced by ATP show that following removal of ATP and additional incubation, a gradual recovery of cyclase activity is observed. This result suggests that under appropriate conditions the cyclase inactivation by ATP is reversible.

Effect of amphotericin B on growth and membrane permeability in Dictyostelium discoideum.

In this study we have determined the effect of the polyene antibiotic amphotericin B on the growth of the eukaryotic microorganism Dictyostelium discoideum. These experiments show that the addition of drug to axenically growing cultures results in an inhibition of growth and cell division. However, with continued incubation, growth is resumed. To determine if the inhibitory effect was due to cell death, the effect of the drug on cell viability was measured. The results showed 10 to 20 times more drug was required to kill cells than to inhibit growth. Since previous studies had indicated that drugs of this type modified cellular permeability, the effect of this drug on osmotic stability of these cells was determined. Results reported in this study show that amphotericin B treatment modifies the cell surface, producing osmotically unstable cells, and that this modification occurs before the onset of cell death and within the same concentration range as used to bring about the inhibition of growth and division. Based on these data it is suggested that the modification in cellular permeability produced by the drug results in the inhibition of growth. This study also reports the results of experiments on the fate of the membrane-damaged cells. These experiments, using radioactive thiourea, showed the restoration of cellular permeability barrier and suggested that the resumption of cell growth occurs after the completion of the repair process.