. Growth of the eel trypanosome, Trypanosoma granulosum, was attempted in five semi‐defined and three defined media. Growth was poor in four semi‐defined and two defined media, but one semi‐defined medium, a modified version of SDM‐79 without MEM F‐14, supported good growth of the trypanosome. In this medium, doubling time was between 1 and 2 days, over 90% of organisms seen in culture after 6 days were trypomastigotes, and 1.8 × 0.1 × 107 trypanosomes ml−1 was achieved in 7 days. When foetal calf serum from the modified SDM‐79 was substituted with insulin to create a fully defined medium, a modest but significant increase in cell numbers occurred compared with controls. In vitro experiments with D, L‐alpha‐difluoromethylomithine (DFMO) showed morphological changes leading to destruction of the trypanosome with increasing concentrations of the drug. A 50‐mM concentration of DFMO inhibited growth by more than 90%, and the IC50 was found to be 16 × 2 mM.
The concentrations of putrescine, spermidine and spermine and the activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAM-D) were investigated in fast muscle subjected to chronic low-frequency electrical stimulation. Both ODC and SAM-D activities increased markedly between 18 and 48 h of stimulation. Changes in enzyme activities were followed by phasic elevations in the concentrations of putrescine, spermidine and spermine. Peak levels were reached first by putrescine at 3-4 days, followed by spermidine at about 9 days and then by spermine at about 11 days. A possible relationship was sought between these events and changes produced in vitro in the phosphorylation pattern of cytoplasmic proteins and the total activity of cyclic AMP-dependent protein kinase. However, during the early stages of stimulation, no prominent changes were seen either in the phosphorylation pattern or in the activity of cyclic AMP-dependent protein kinase. These characteristics changed significantly at a later stage (by 12 days of stimulation) and became indistinguishable from those of slow muscle by 3 to 4 weeks of stimulation.
Polyamines are important in the growth, division and differentiation of many cell types, including trypanosomes. The ultrastructure of culture forms of Trypanosoma granulosum was examined following growth in a modified semidefined medium (controls) and in the same medium with added polyamine biosynthesis inhibitors (DFMO, MGBG and Berenil). Untreated trypanosomes had ultrastructural features in common with other cultured flagellates. Those treated with DFMO were generally more rounded in contour. Cytoplasmic vacuoles and swollen mitochondrial membranes suggested osmotic imbalance, perhaps resulting from compromised membrane integrity. An anticlockwise arm dividing subtubule B of the peripheral doublets of the flagellum was noted in some trypanosomes treated with 20 mM DFMO. Vacuolation was also induced by MGBG and flagellar division occurred more frequently at 0.2 mM than in controls. With 1 mM MGBG, mitochondria were difficult to discern and kinetoplasts were disrupted. With 0.2 mM Berenil, vacuoles, swollen mitochondria, membranous whorls, additional microtubules underlying subpellicular tubules and disaggregated kinetoplasts were noted. With 1 mM Berenil, much of the cell structure was destroyed, although the pellicle, flagellum and paraxial rod remained intact. The present study illustrates the importance of hnctional polyamine synthetic pathways for the integrity of membranes, mitochondria, kinetoplasts and possibly microtubules in cultured T. granulosum.
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