Differentiation-Dependent Decline of DNA Synthetic Activities in Naegleria gruberi

SYNOPSIS. DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [³H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transforms, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [³H]thymidine and [³H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at ∼ 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts ∼ 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase.     

The Effects of Selected Chemical Agents on the Amoeba-Flagellate Transformation in Naegleria gruberi*

SYNOPSIS. Amoeboid Naegleria gruberi grown on an agar surface were induced to transform synchronously into flagellates by changing the pH of the environment from 3.8 to 7.2. Flagellates started to appear 70 min after stimulation, reached the 90% level within the next 30–40 min, and gradually reverted to the amoeboid form in the next several hrs. The presence of previously induced cells did not influence normal induction of transformation, indicating that no interaction took place during transformation. Inhibitors of oxidative phosphorylation (DNP and cyanide), of protein synthesis (puromycin and cycloheximide), and of RNA synthesis (actinomycin D), delayed or blocked the transformation, suggesting that RNA and protein synthesis are required. Because the flagellated stage is shortened by puromycin treatment, protein synthesis appears to be linked to the duration of the flagellated stage.     

Deposition of Basic Fibroblast Growth Factor on Surface of Epidermal Melanocytes Suggesting the Stromal Control of Epidermal Pigmentation

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.