PubMed Health⌕ Search

Biomedical subjects

E F Rossomando

Publications and source records attributed to E F Rossomando.

At least 73 records · Page 4Linked to original sources
PubMed1978

Effect of hadacidin on growth and adenylosuccinate synthetase activity of Dictyostelium discoideum.

The growth of the eucaryotic microorganism Dictyostelium discoideum in liquid culture was completely inhibited by the aspartic acid analog hadacidin (N-formylhydroxyamino-acetic acid). Growth arrest occurred both in chemically defined medium and in complex growth medium containing aspartic acid and AMP precursors such as adenine and adenosine. Although these compounds could not overcome the effect of hadacidin, growth was restored if cells were washed and resuspended in fresh growth medium. Additional experiments showed that D. discoideum contains adenylosuccinate synthetase, the enzyme which catalyzes the synthesis of adenylosuccinate from IMP, aspartic acid, and GTP in the de novo biosynthesis of purines. A partially purified preparation of this enzyme was obtained, and the effect of hadacidin on its activity was studied. We found that maximum inhibition of the D. discoideum activity occurs at a ratio of aspartic acid to hadacidin of 5:1, suggesting that the affinity of the drug for this enzyme is less than for the enzyme from rabbit muscle and plants but greater than for that from Escherichia coli. The effect of the drug can be overcome by a 10-fold excess of aspartic acid, suggesting that the drug acts as a competitive inhibitor. A comparison of the adenylosuccinate synthetase activity levels at various stages of growth showed that its specific activity decreases about 60% as cells enter the stationary growth phase, and decreases about 75% after starvation for 2 h. Further studies showed that in cells treated with hadacidin the rate of uptake of exogenous nutrients is reduced about 75% and that these cells are more resistant to rupture by osmotic shock. While the results of this study are consistent with the proposal that growth arrest is contingent upon inhibition of adenylosuccinate synthetase activity, they also suggest that, as a consequence of this inhibition, some physiological properties of the cell have been altered.

Adenylosuccinate Synthase↗
PubMed1978

Protease secretion during onset of development in Dictyostelium discoideum.

At the onset of development, the single cells of the eukaryotic micro-organism Dictyostelium discoideum secrete proteolytic activity which can be assayed using the insoluble substrate remazolbrilliant blue hide. The activity is not secreted by exponentially growing cells, but does appear extracellularly at the onset of the stationary growth phase. When growth phase cells are resuspended in non-nutrient buffer, proteolytic activity begins to appear outside the cells. It accumulates in the buffer at a rate similar to that observed for 2 glycosidases of lysosomal origin and reaches a maximum after about 2 h of incubation. After 3--4 h incubation, centrifugation of the non-nutrient buffer removes the cells, producing a supernatant which we refer to as conditioned medium. Subsequent experiments with conditioned medium showed: (a) its incubation with purified plasma membranes results in the release of polypeptides which can be recovered and, when displayed on polyacrylamide gels, can be shown to be stage specific; and (b) that conditioned medium can decrease the rate of detachment of cells from a collagen substratum. Both effects can be prevented by the addition of remazolbrilliant blue hide suggesting that they are due to proteolytic activity present in the conditioned medium. Finally, we were able to show that conditioned medium contains components which, when spread over the bottom of plastic Petri dishes, enhance the rate of multicellular structure formation. Additional studies showed that this effect of conditioned medium could also be brought about by components which remained behind on uncoated plastic dishes after the removal of a D. discoideum cell layer. These data may be accommodated to a model in which the protease secreted during the onset of development acts on the cell membrane releasing components which coat the substratum and facilitate migration and multicellular structure formation.

Cell Membrane↗
PubMed1977

Synthesis of a mannosyl phosphoryl polyprenol by the cellular slime mold Dictyostelium discoideum.

A membrane fraction isolated from the cellular slime mold Dictyostelium discoideum was incubated with GDP-[14C]mannose and found to catalyze the incorporation of [14C]mannose into an endogenous acceptor to yield a product with the chemical and chromatographic properties of a polyprenol phosphate sugar derivative. These results suggest that D. discoideum can synthesize a mannosyl phosphoryl polyprenol.

Dictyostelium↗
PubMed1977

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.

Amphotericin B↗
PubMed1976

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.

Adenosine↗
PubMed1976

DNA polymerase of Dictyostelium discoideum.

Only one molecular weight species of DNA polymerase was found in different developmental stages of the eukaryotic microorganism Dictyostelium discoideum. The molecular weight of this DNA polymerase is estimated to be about 127 000 by sucrose gradient centrifugation. The enzyme is present in all stages of growth and development, including dormant spores. All DNA polymerase activity is lost upon incubation of the crude extract with N-ethylmaleimide. The reaction properties, molecular weight and N-ethylmaleimide sensitivity of the D. discoideum DNA polymerase are similar to those of the DNA polymerase-alpha from mammalian sources.

Binding Sites↗
PubMed1976

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.

Amphotericin B↗
PubMed1973

A 5'-Adenosine Monophosphate-Dependent Adenylate Cyclase and an Adenosine 3':5'-Cyclic Monophosphate-Dependent Adenosine Triphosphate Pyrophosphohydrolase in Dictyostelium discoideum.

Cell aggregation in Dictyostelium discoideum appears to involve the production and detection of 3':5'-cyclic AMP. Two pertinent catalytic activities have been studied in concentrates from D. discoideum purified 50- to 100-fold. They are (i) adenylate cyclase and (ii) ATP pyrophosphohydrolase. ATP pyrophosphohydrolase activity converts ATP to 5'-AMP and pyrophosphate. The presence of 5'-AMP is an absolute requirement for adenylate cyclase activity. The presence of 3':5'-cyclic AMP is an absolute requirement for ATP pyrophosphohydrolase activity. Both activations, particularly that of ATP pyrophosphohydrolase, show narrow ranges of specificity and display significant cooperativities.

Journal Article↗