Phospholipase D in Tetrahymena: activity and significance

We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture.     

Methotrexate permits a limited number of cell doublings and affects mitochondrial substructure inTetrahymena

Summary Methotrexate (MTX), a folic acid analogue, is used in cancer chemotherapy in low or high doses. Addition of MTX to proliferatingTetrahymena cultures revealed that, irrespective of the concentration of MTX (1–50 mM), the cells doubled once during a 6-hour exposure and twice during a 24-hour exposure; the normal generation time is 3 hours. MTX had a dose-dependent effect on the maximal number of cell doublings reached after 72 hours, thus 5 1/2 and 3 1/2 doublings were found in 1 and 10 mM MTX, respectively. Massive cell death was observed after a 4-day exposure. At all concentrations of MTX, the rate of endocytosis was unaffected initially but decreased to 60% of the control value after 24 hours. Conspicuously, small refractive, or electron dense, granules accumulated in MTX-treated cells; these granules are presumably lysosomes accumulating MTX for defecation. The mitochondrial substructure became altered in MTX-treated cells; after a 24-hour exposure to MTX the mitochondria were almost depleted of tubules. This reduction of the number of mitochondrial tubules occurred gradually, apparently correlated with division of the organelles. Recovery was also studied of cells exposed for 1 or 3 hours to 10 mM MTX. During the first 24 hours after removal of MTX, the cells were affected in a manner similar to that described for cells exposed continuously to MTX, also with respect to the altered substructure of mitochondria; however, the recovering cells resumed the normal rate of cell proliferation after 4 cell doublings. These longlasting effects, of even a short exposure ofTetrahymena to MTX, may in part explain the cause of the severe side effects accompanying MTX chemotherapy.     

Human cytokines interleukin (IL)-3 and IL-6 affect the growth and insulin binding of the unicellular organismTetrahymena

Interleukin (IL)-3 and IL-6 significantly increase the growth rate of the unicellular organism,Tetrahymena. The effect elicited by IL-3 is long lasting as it was also detectable after 20 generations. Effect of IL-6 was detectable as long as the substance was present in the cell culture. Pretreatment with IL-3 did not enhance the proliferative response to subsequent IL-3 treatment, but the second exposure to IL-3 considerably depressed the active proliferation ofTetrahymenacells. However, a positive ‘priming effect’ elicited by IL-6 resulted in an increased growth rate following repeated IL-6 stimulation. Insulin binding to the plasma membrane ofTetrahymenawas increased by IL-6 but not by IL-3 after 24 hours, and this enhancement appeared even after one hour incubation. If the cells were pretreated with insulin, IL-6 did not influence insulin binding, while an inhibition by IL-3 was observed. These results direct attention to the similarities of actions induced by IL-3 and IL-6 at different levels of phylogeny probably due to the presence of cytokine receptor-like structures on this unicellular organism.     

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.     

Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

Effect of medium copper concentration on the growth,uptake and intracellular balance of copper and zinc in Menkes' and normal control cells

The precise nature of the variation in cellular copper load against medium copper concentration is defined using a comprehensive logarithmically incremented series of medium copper concentrations ranging from low levels (4.8 p.p.b.) through normal to toxic levels (40 p.p.m.) in which fibroblasts were grown followed by determination of intracellular content. Menkes' fibroblasts showed an unexpected plateau region of stable intracellular copper content against a change in medium concentration of over 100-fold, albeit only when sufficient copper was present in the medium (0.08–8.0 p.p.m.). Thus, Menkes' cells are clearly capable of balancing uptake/efflux providing copper availability allows. Simultaneous analysis of cellular copper and zinc load at various medium copper concentrations shows an indistinguishable intracellular copper:zinc ratio between the two cell lines. The nature of non-labeled copper uptake by fibroblasts over a 40 min and 7 day period is reported. During the 40 min period copper uptake (20 p.p.m.) was essentially the same in both cell lines. However, copper absorbed was superimposed upon large pre-existing copper pools in the case of Menkes' cells only. Advantages of techniques determining non-labeled copper in copper uptake/efflux experiments are discussed in the light of these results. Fibroblast growth studies showed that, compared with normal cells, Menkes' cells are significantly (P < 0.01) more growth sensitive to extended exposure to low copper concentrations. Thus, Menkes' disease appears to be not only a result of copper maldistribution but also a direct result of an inability of Menkes' cells to function normally in low copper environments.     

Nutritional stress in Tetrahymena relieved by addition of hemin or phospholipids

Summary The ciliate Tetrahymena thermophila was grown in a synthetic nutrient medium at various amino acid concentrations. Before the beginning of the experiments the cells were starved for 4 h in a pH buffer. They were inoculated at an initial density of only 250 cells per ml. Under these conditions the cells grew and multiplied at only the two highest amino acid concentrations used. Hemin or phospholipids were found to stimulate cell growth at the lower amino acid concentrations. The mechanism behind this stimulatory effect is unknown, but may be connected with the maintenance of an adequate energy flow under adverse conditions. These additions represent an improvement of the synthetic medium for Tetrahymena.Abbreviations PPYS proteose peptone, yeast extract, and salts medium     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

SHORT COMMUNICATIONSTEROID HORMONE (HYDROCORTISONE, OESTRADIOL AND TESTOSTERONE) UPTAKE, STORAGE OR INDUCED SYNTHESIS INTETRAHYMENA

After cyclodextrin-coated 10⁻⁶ steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P₄₅₀aromatase.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited, at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a Junction of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

Phosphonoacetic Acid: Effect on and Disposition by Tetrahymena1

The antiviral agent phosphonoacetic acid inhibits growth of Tetrahymena thermophila at concentrations comparable to those inhibiting growth of other eukaryotic cells, with 50% inhibition at 0.5 mM phosphonoacetic acid. The compound is cytotoxk to Tetrahymena at concentrations greater than 2.0 mM. When a culture of Tetrahymena the growth of which was totally inhibited by 2.0 mM phosphonoacetic acid was diluted with fresh medium, growth resumed in an exponential, rather than synchronous, fashion. [2–¹⁴C]phosphonoacetic acid is not metabolized by Tetrahymena.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

Arrested development in Fucus spiralis (Phaeophyceae) germlings exposed to copper

Exposure of Fucus spiralis germlings to precise copper concentrations (0 to 844?nM?Cu²⁺) in chemically defined medium demonstrated a relationship between ultrastructural changes and growth retardation with increasing copper concentration. Electron-translucent vesicles, present in ova, which normally disappear after fertilization, accumulated in germlings exposed to Cu²⁺ above 10.6?nM, suggesting that copper may inhibit a metabolic pathway involved in cell wall formation which is initiated by fertilization. No membrane damage was observed during the exposure period. During a post-exposure period in copper-free medium, recovery occurred (rhizoid extension, apical hair formation) in germlings previously exposed to concentrations below 106?nM?Cu²⁺ and electron-translucent vesicles became granular and disappeared. It is proposed that the electron-translucent vesicles contain a cell wall precursor and that copper inhibits its incorporation into the cell wall, preventing growth and development of the zygote.     

Inhibition by Theophylline of Phagocytosis in Acanthamoeba castellanii*

SYNOPSIS. Theophylline inhibited the phagocytosis of latex beads by Acanthamoeba castellanii (Neff). The cells recovered the ability to engulf beads after 1–2 hr of exposure to theophylline. Cells which have been exposed to 25 mM theophylline for a period of inhibition and recovery were not inhibited further by incubation with a fresh medium containing the same concentration of theophylline. However, the medium in which the cells recovered was as effective as a fresh medium in inhibiting phagocytosis in a fresh batch of cells, suggesting that the development of insensitivity to theophylline inhibition resides with the cells themselves. Dibutyryl cyclic AMP also inhibited bead uptake.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural Changes and Their Reversibility*

SYNOPSIS. Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.     

Adaptation of a Saccharomyces cerevisiae strain to high copper concentrations

A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.     

Effect of Biogenic Amines on Phagocytosis in Tetrahymena thermophila1

Stimulation of phagocytosis by serotonin and catecholamincs in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being ∼0.1 to 1.0 μM. The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the β- and α-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis. A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena. Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine. Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.     

Use of an industrial grade medium and medium enhancing effects on high cell density CO fermentation by Eubacterium limosum KIST612

Studies were made on the composition of the growth medium to increase the cell concentration in a cell-recycled continuous culture (Eubacterium limosum KIST612) with carbon monoxide as a sole energy source using phosphate-buffered basal medium (PBBM) and modified PBBM. One of major limiting factors in PBBM might be nitrogen during the high cell density culture. This limitation could be overcome by increasing of inorganic nitrogen or yeast extract concentration in the medium. Anaerobic digester fluid, which could replace the organic nitrogen in PBBM, was used to develop an industrial grade medium for conversion of CO to multi-carbon compound.