The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.     

Kinetics of growth of the ciliate Tetrahymena pyriformis on Escherichia coli

The growth of the ciliate Tetrahymena pyriformis on non-growing Escherichia coli has been studied by following the time courses of population densities and protozoan mean cell volume in batch cultures. Viable, non-encysted protozoa always stopped feeding before the bacterial density was reduced to zero and non-feeding ciliates tended to swim faster than feeding ciliates. In addition, the number of bacteria and other particles of bacterial size consumed in the formation of one new ciliate, when averaged over the lag and reproductive phases of a culture, declined toward a limiting value of about 1.6 x 10(4) particles per ciliate as the initial density of such particles was increased.     

The influence of the EGF on proliferative signal transduction in ciliate Tetrahymena pyriformis

It has been shown that the transduction of the proliferative signal induced by EGF in ciliata Tetrahymena pyriformis cells is not connected with autophosphorylation of the receptor tyrosine kinases. The results obtained indicate that EGF in ciliata cells initiates the mitogenic pathway including the membrane proteins of the tyrosine kinases-like type (without tyrosine phosphorylation sites), adenylate cyclase, tyrosine- and Ca2+ -dependent ERK-like kinases.     

Continuous culture of the ciliate Tetrahymena pyriformis on Escherichia coli

The ciliated protozoan Tetrahymena pyriformis was grown in a chemostat fed with a culture of Escherichia coli overflowing from another chemostat. Densities of the protozoan and bacterial populations, mean volume of protozoan cells, yields of protozoan volumes and numbers, and filtering rates of protozoans per cell and per unit volume of biomaterial were determined at five different dilution rates. The data obtained supplement other data already available for the popular test organism T. pyriformis, and they are also comparable with data available for related ciliates.     

Chemotactic effect of odorants and tastants on the ciliate Tetrahymena pyriformis

Naturally occurring aroma compounds are able to elicit physiological and migratory responses such as chemotaxis even at nano to femtomolar concentrations in organisms at different levels of phylogeny. Despite the amazing chemical variety of these substances the apparatus by which they can be detected i.e. the chemosensory receptors and the signaling pathways seem to be rather uniform and evolutionary well-conserved. The intracellular signaling process is supposed to be mediated by either cAMP or inositol 1,4,5-trisphosphate. The present work aimed to investigate the chemotactic behavior of 11 odorants that occur naturally in foods and are also used by the industry as additives, on the eukaryotic ciliate Tetrahymena pyriformis. Intracellular signaling pathways that might be activated by these compounds were also investigated. Activation of the phospholipase C (PLC) was measured by FACS and the stimulation of inositol-1,4,5-trisphosphate 3-kinases (IP3K) was measured using two specific inhibitors, wortmannin and LY294002. The strongest chemoattractant character was observed for isoamyl acetate (10(?6) M), propyl isobutyrate (10(?8) M), isobutyl propionate (10(?6) M). The strongest repellent action was exerted by benzyl acetate (10(?8) M), furfuryl thioacetate (10(?12) M). Our results suggest that Tetrahymena responds in a very sensitive way to slight changes in the molecular structure. According to our study, tracer amounts of solvents do not contribute significantly to the chemotactic profile of the respective odorants. No significant activation of PLC or PI3K could be observed following stimulation with attractant odorants which implies that some other pathways may be involved, hence further investigation is needed.     

Chemotactic effect of odorants and tastants on the ciliate Tetrahymena pyriformis

Naturally occurring aroma compounds are able to elicit physiological and migratory responses such as chemotaxis even at nano to femtomolar concentrations in organisms at different levels of phylogeny. Despite the amazing chemical variety of these substances the apparatus by which they can be detected i.e. the chemosensory receptors and the signaling pathways seem to be rather uniform and evolutionary well-conserved. The intracellular signaling process is supposed to be mediated by either cAMP or inositol 1,4,5-trisphosphate. The present work aimed to investigate the chemotactic behavior of 11 odorants that occur naturally in foods and are also used by the industry as additives, on the eukaryotic ciliate Tetrahymena pyriformis. Intracellular signaling pathways that might be activated by these compounds were also investigated. Activation of the phospholipase C (PLC) was measured by FACS and the stimulation of inositol-1,4,5-trisphosphate 3-kinases (IP3K) was measured using two specific inhibitors, wortmannin and LY294002. The strongest chemoattractant character was observed for isoamyl acetate (10^?6 M), propyl isobutyrate (10^?8 M), isobutyl propionate (10^?6 M). The strongest repellent action was exerted by benzyl acetate (10^?8 M), furfuryl thioacetate (10^?12 M). Our results suggest that Tetrahymena responds in a very sensitive way to slight changes in the molecular structure. According to our study, tracer amounts of solvents do not contribute significantly to the chemotactic profile of the respective odorants. No significant activation of PLC or PI3K could be observed following stimulation with attractant odorants which implies that some other pathways may be involved, hence further investigation is needed.     

RNA synthesis and cell division in cold-synchronized cells of Tetrahymena pyriformis

Cells of Tetrahymena pyriformis have been cold-synchronized using a repetitive cycle of six, two-hour cold shocks (9.5°C) alternating with decreasing periods (60–30 minutes) at 28°C. This system gives a maximum division index of 70–80% occurring at 90 minutes from the end of the last synchronizing cold-treatment (EC). Examination of the division sensitivity of these cells to actinomycin D applied continuously at ten-minute intervals from EC reveals that division is essentially blocked until approximately 40 minutes past EC, after which a rapid decrease is sensitivity to the inhibitor occurs. Coinciding with this period of high sensitivity is the occurrence of a peak of C¹⁴ uridine incorporation at 40 minutes past EC. Inhibition of this peak is correlated with an inhibition of division, whereas strong inhibition of RNA synthesis beyond 60 minutes past EC has little effect on division activity. The similarity of these findings with those of the heat-synchronized system is discussed with the suggestion that both heat- and cold-synchronizing treatments result in the synchronous resynthesis of a division-associated fraction of RNA.     

Polyterpenoids as cholesterol and tetrahymanol surrogates in the ciliate Tetrahymena pyriformis

The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols. The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect. In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T. pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids. Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids. No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability. Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T. pyriformis cells.     

Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

The arsenic metabolism in different biological organisms has been studied extensively. However, little is known about protozoa. Herein, we investigated the cell stress responses of the freshwater ciliate Tetrahymena pyriformis to arsenate toxicity. An acute toxicity assay revealed an 18-h EC(50) arsenate concentration of ca. 40 μM, which caused significant changes in the cell shape, growth and organism mobility. Whereas, under exposure to 30 μM arsenate, T. pyriformis could grow reasonably well, indicating a certain resistance of this organism. Arsenic speciation analysis revealed that 94-98% of the total arsenate in cells of T. pyriformis could be transformed to monomethylarsonic acid, dimethylarsinic acid and a small proportion of arsenite after 18 h of arsenate exposure, thus indicating the major detoxification pathway by arsenic oxidation/reduction and biomethylation. Finally, comparative proteomic analysis unveiled significant changes in the expression of multiple proteins involved in anti-oxidation, sugar and energy metabolism, proteolysis, and signal transduction. Our results revealed multiple pathways of arsenate detoxification in T. pyriformis, and indicated that protozoa may play important roles in the biogeochemical cycles of arsenic.     

Promotion of gas bubble formation by ingested nuclei in the ciliate,Tetrahymena pyriformis

Cells of the ciliateTetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1−0.5×10⁷ Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.     

Promotion of gas bubble formation by ingested nuclei in the ciliate, Tetrahymena pyriformis

Cells of the ciliate Tetrahymena pyriformis were suspended with carmine or graphite particles or with Halobacterium gas vesicles, all of which promote bubble formation in aqueous suspensions when tested with 10 atm and above (0.1-0.5 X 10(7) Pa) (carmine and graphite) or 25 atm and above (gas vesicles) of nitrogen supersaturations. All three particles were ingested, but only the gas vesicles promoted intracellular gas bubble formation if the cells containing them were nitrogen or methane saturated in a slow stepwise fashion prior to rapid decompression. Cell rupture did not occur until gas saturation pressures greater than 25 atm were used; this suggests that the ciliate pellicle and cytoplasm cannot resist the mechanical forces of an expanding gas phase induced by decompression from between 25 and 50 atm and thus provides an estimate of the physical strength of these cellular components. The inability of the ingested carmine, graphite, and collapsed gas vesicles to induce intracellular gas bubble formation suggests that the phagocytic process somehow altered them. This procedure may thus provide a tool for the study of early events in the digestive processes of ciliates.