Köln, Universiẗat, Diss., 2001.

Köln, University, Diss., 2003.

Heidelberg, Universiẗat, Diss., 2003. / Dateien im PDF-Format.

Köln, University, Diss., 2003.

Köln, University, Diss., 2004.

The effect of cyclic adenosine 3', 5' monophosphate (cyclic AMP ) on ionic movement across the cell-membrane of the cellular slime mold Dictyostelium discoideum, strains V-12 and NC4, has been studied by using radioisotopes, Na²² and Ca⁴⁵. D. discoideum was grown on water agar associated with autoclaved radioactive E. coli 281.The experimental amoebae were centrifuged to free them from bacteria. Cyclic AMP was added to the experimental culture every 10 or 20 minutes. The results showed that cyclic AMP has a significant effect on calcium ion movement across the membrane both in the pre-aggregative amoeboid stage and during the aggregating phase. The movement of sodium ion was changed only during aggregation. The addition of phosphodiesterase the enzyme -which inactives cyclic AMP by converting it to 5' AMP, showed no effect on ion transport either in the pre-aggregative stage or the aggregating stage. The ionic contents of sodium, potassium, and calcium, were measured at the slug stage. The relationship of calcium ion and cyclic AMP to amoeboid movement was compared with the role of cyclic AMP in muscular contraction and other function. / Science, Faculty of / Zoology, Department of / Graduate

No description available.

The effects of stalk cell differentiation inducing factor (DIF) on stalk cell induction from vegetative cells and prestalk cells of Dictyostelium discoideum were investigated. It was found that the major DIF component DIF-1 is a poor inducer of stalk cell formation from prestalk cells, but a minor component, DIF-2 is a more important inducer for the conversion of prestalk to stalk cells. Evidence is presented that DIF-2 synergizes the activity of the other DIF components for prestalk to stalk cell conversion. In contrast DIF-5 inhibits the activity of DIF-1 and DIF-3/4. A model is proposed to explain those results. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The membrane bound alkaline phosphatase activity' in vegetative cells of Dictydstelium discdideum was found to exhibit a 8 to 10 fold increase in specific activity when incubated at 50°C. This activation was reversed on transfer of the preparation to 0°C. Similar activation of the vegetative enzyme was achieved by dialysing the crude membrane preparation, suggesting the removal of a low molecular weight inhibitor. Alkaline phosphatase solubilized from the membranes using Triton X-100 was similarly activated by 50°C treatment or dialysis and the 50°C activation was reversed by incubation at 0°C. Both the dialysed vegetative membrane and the dialysed Triton X-100 extract were inhibited by the addition of concentrated dialysate. This inhibition could be relieved by subsequent dialysis. A 620 fold purified alkaline phosphatase preparation was obtained from crude vegetative membranes by affinity and ion exchange chromatography after solubilization of the enzyme using Triton X-100-5'-nucleotidase activity copurified along with the alkaline phosphatase activity in all the fractionation steps employed. The membrane bound 5'-nucleotidase activity was not activated either by incubation at 50°C or by dialysis and the activity was far less stable than the alkaline phosphatase. However, the Triton X-100 extracted 5'-nucleotidase was activated by dialysis to the same extent as the alkaline phosphatase and both activities in the partially purified preparation were equally inhibited by the dialysates from vegetative membranes. These results suggest that both alkaline phosphatase and 5'-nucleotidase are due to a single protein but the interaction of the two substrates, AMP and pNPP, with the enzyme are different and are markedly influenced by conformational changes induced by the inhibitor. Unlike the vegetative enzyme, the alkaline phosphatase activity in the culminating membrane was not markedly activated by incubation at 50°C or by dialysis. Since the vegetative enzyme was activated by both treatments to levels similar to those found in culminating cells, it was proposed that the developmental increase in alkaline phosphatase in D.discoideum was due to the unmasking of already existing enzyme by the removal of inhibitor. However, the alkaline phosphatase activity of culminating cells differed from that of vegetative cells in chromatography on DEAE-Sephacel and conA-Sepharose and it was less stable in low concentrations of Tris-Cl and in SDS. In contrast, the culminating enzyme was more stable in high concentrations of Tris-Cl. These results suggest that the vegetative enzyme is modified during development. This modification appears to be slight, since the vegetative and culminating enzymes migrate identically in SDS polyacrylamide electrophoretic gels and the enzymes from the two developmental stages were similar in pH optima, in inhibition by phosphate and in inhibition by concentrated dialysate. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The expression of a mammalian ras gene analog was previously found by Reymond et al. to be developmentally regulated in Dictyostelium discoideum using Northern analysis of strain AX-3 RNA (1984, Cell 39;141) and by Pawson et al. using specific immunoprecipitation of in vivo synthesized proteins from strain V12M2 (1985, Mol. Cell Biol. 5;33). Due to differences in the results of the two studies, it was decided to further examine ras expression by applying both protein and RNA techniques to a single strain of D.discoideum, V12M2. RNA samples from strain V12M2 cells at different stages of development were analyzed using Northern blotting. The same RNAs were translated in vitro, and the ras proteins synthesized were immunoprecipitated and analysed by polyacrylamide gel electrophoresis. In agreement with the findings of Reymond et al. (1984, Cell 39;141), Northern analysis with the cDNA ras probe revealed that the highest levels of the 1.2 and 0.9 kb ras mRNAs were present in the total RNA of V12M2 cells at the pseudoplasmodial stage of development, and very little ras mRNA was present in early developing cells. In contrast to the Northern analysis the greatest amount of ras protein was in vitro translated from the RNA of vegetative and 2 hour cells. Hence this work confirms in a single strain of Dictyostelium that the greatest amount of ras protein is synthesized at those developmental stages that contained the lowest levels of mRNA detectable by the cDNA probe. Possible reasons for this phenomena are discussed. In vitro RNA translation was also used to study the relationship between the two ras proteins of 23 and 24 kd. The proteins did not appear to be derived from one another by degradation or by post-translational modification. This result suggested that the two ras proteins of strain V12M2 must be derived from two different mRNAs. High stringency Southern blots of AX-3 DNA showed the expected restriction fragments detected by Reymond et al. (1984, Cell 39;141) . Low stringency blots showed three faint additional restriction fragments in Eco RI digests of AX-3 DNA. No additional restriction fragments were generated by an Eco RI-Bg1 II digest, but two of the three faint bands were smaller. This suggested that at least two of the Eco RI ras fragments are non-contiguous, and hence two to three ras genes may be present in addition to the one characterized by Reymond et al. (1984, Cell 39;141). All Northern and Southern bolts were probed with antisense RNA probes in order to gain greater sensitivity of detection as described by Cox et al. (1984, Dev. Biol. 101;485). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate