How applicable is the general adaptation syndrome to the unicellular Tetrahymena?

Hormone receptors, hormones and signal transduction pathways characteristic of higher vertebrates can be observed also in the unicellular Tetrahymena. Previous work showed that stress conditions (starvation, high temperature, high salt concentration, formaldehyde or alcohol treatment) elevated the intracellular level of four hormones (ACTH, endorphin, serotonin and T₃). Here, the effect of other stressors (CuSO4 poisoning, tryptophan hydroxylase inhibitor parachlorphenylalanine (PCPA) treatment) on the same and other hormones (epinephrine, insulin, histamine) was studied, using immunocytochemistry and flow cytometric analysis. It was found, that each effect increased the intracellular hormone contents, but some hormones (histamine, T₃) were less reactive. Insulin—which is a life‐saving factor for Tetrahymena—itself provoked elevation of hormone amounts in association with a stressor, further increased the level of hormones. It was concluded that the ancestor of Selye's General Adaptation Syndrome (GAS) can be found already at unicellular level, and this possibly has a life saving function. Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of stress and stress hormones on the hormone (insulin) binding of Tetrahymena

The unicellular Tetrahymena pyriformis GL produce, store and secrete vertebrate‐like hormones. In earlier experiments the effect of different stressors on the hormone levels of Tetrahymena was studied and an elevation of these was found. In the present experiments the hormone binding was investigated, using flow cytometric method. FITC‐insulin binding was elevated after concentrated (5, 10, or 20 mg ml^?1) NaCl or 0.01%, 0.1%, or 0.05% formaldehyde treatment, or after thermal stress (37°C). Serotonin given together with NaCl increased and together with formaldehyde decreased the binding. Histamine always decreased the binding and insulin was indifferent. Four hours after osmotic stress, hormone binding significantly decreased and this was not influenced by hormones. However, 4 h after formaldehyde stress the binding elevated and this was further increased by repeated hormone treatments. The results show that the stress in Tetrahymena provokes an activation of its hormonal system (hormone production and binding), which is differently influenced by exogeneously given hormones. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of concanavalin A (Con-A) on the hormone production of the unicellular Tetrahymena and the immune cells of the rat. A comparative study

Tetrahymena populations were treated with 10(-15) g ml(-1) or 10(-6) g ml(-1) concanavalin-A (Con-A) in tryptone-yeast medium for 1 h. Rat peritoneal immune cells (mast cells, lymphocytes, monocyte-granulocyte group) were also treated with 10(-6) g ml(-1) Con-A, for 1 h. The cells' hormone (ACTH, histamine, serotonin, endorphin, triiodothyronine (T(3))) content was measured by using immunocytochemistry and flow cytometry. The extremely low dose of Con-A universally and significantly elevated the hormone contents, while the result of higher dose was uncertain. In the immune cells, Con-A significantly decreased the ACTH level in each cell type and histamine level in mast cells. The results demonstrate the very high sensitivity of Tetrahymena receptors for a non-hormone (lectin) molecule, which can bind to the insulin receptors and mimics the effect of insulin. The results also show that Tetrahymena receptors are more sensitive to lower concentrations of molecules than to higher ones. The universal hormone-production stimulating effect of Con-A-which is observed in Tetrahymena-is specified in rat.     

Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions

Melatonin content in the cellular fraction and medium of Tetrahymena pyriformis GL cultures was measured at different time points of light and dark exposures. Tetrahymena produced, stored and secreted immunoreactive melatonin, which in displacement and HPLC studies, behaved like synthetic melatonin. There was not a continuous secretion of melatonin produced by the cells. In contrast to this, storage of melatonin was observed, which was more expressed in dark conditions. Prolonged light exposure suppressed melatonin production and secretion alike, however it did not block it completely.     

Effect of vasoactive peptides on Tetrahymena. chemotactic properties of endothelins (ET-1, ET-2, ET-3, fragment 11-21 of ET-1 and big endothelin-1): a short-term inducible signalling mechanism of chemotaxis

The unicellular Tetrahymena is a sensitive model for the study of chemotaxis induced by endothelins. In short-term chemotactic responses, ET-2 and ET-3 were chemorepellent, compared to the referent control (culture medium) and chemoattractant, ET-1. These differences suggest that the change of some aromatic residues in the loop region (residues 5-9) of the ET-1 abrogates its chemoattractant character. The response of Tetrahymena is highly selective, since substitution of two amino acids are enough to cause this alteration in (behavioural) response. Such a change seems to be more important than the loss of the entire first 10 amino acids (in ET-1 fragment 11-21), since after this it acquires some chemoattractive effect of ET-1. Selection with ET-3 rigorously stimulated the cell's responsiveness to the medium, this ability was abolished by the repeated ET-3 treatment. Big endothelin-1 was repellent in all concentrations. These experiments demonstrate the very sensitive discriminating capacity of chemotactic responsiveness at a low level of phylogeny. Chemotactic selection with endothelins underlines the possibility that there may be separate mechanisms responsible for the short-term chemotactic responses and the long-lasting effects of chemotactic selection.     

Effects of steroid hormones on five functional parameters of Tetrahymena: evolutionary conclusions

The unicellular Tetrahymena pyriformis was studied for chemotaxis, chemotactic selection, phagocytosis, growth and body shape changes in the presence of water soluble (beta-cyclodextrin-coupled) steroid hormones (testosterone, estradiol, progesterone, hydrocortisone and dexamethasone). Testosterone was chemoattractant over a wide range of concentrations, while progesterone and dexamethasone were active only at one concentration (10(-5) and 10(-6) mg ml(-1) respectively) and were either neutral or repellent at other concentrations. Hydrocortisone and estradiol were unambiguously chemorepellent. Chemotactic selection enhanced the effect of testosterone and estradiol, while in the case of hydrocortisone the action was reversed. The other parameters were mildly influenced by the steroid hormones. The results call attention to the fine molecular recognition capacity of Tetrahymena and to the possible rapid effects of steroid hormones at membrane receptors at a very low evolutionary eukaryotic level.     

Induction of hormone receptor formation in the unicellular tetrahymena

Studies based on treatment with antibodies to thyrotropic hormone, luteotropic hormone, growth hormone or adrenocorticotropic hormone have shown that although the unicellular Tetrahymena does not possesssui generis receptors to all polypeptide hormones, such binding structures may arise, or become established in the membrane of the unicellular Tetrahymena in the presence of exogenous hormone. The Tetrahymena subjected to hormonal imprinting still contained an increased amount of hormone after six generation changes, which suggested that either hormone production had been induced by treatment, or the internalized hormone had not been degraded intracellularly. Thus the role of hormonal imprinting in receptor formation has also been substantiated by the immunocytochemical approach used in the present study.     

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species(ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membranebound vacuoles characteristic of autophagy followed by autophagic cell death(referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.     

Induction of digoxin-like material production, and the digoxin binding in the unicellular organism Tetrahymena by digitoxin.

Thin layer chromatographic, and laser-confocal microscopic analyses with a monoclonal antibody to digoxin also displaying high affinity to digoxigenin, were used to determine the presence and localization of cardioactive glycosides. Tetrahymena pyriformis was found to possess digitoxigenin-like material, but digoxin, digitoxin, digoxigenin, gitoxin and lanatoside C were not detected. Digitoxin treatment elicited the appearance of a digoxin-like material in the progeny generations. Digoxin was taken up by untreated Tetrahymena, especially strongly 24 h after digitoxin treatment. While the cardenolide was localized in vesicles of the cell body in untreated Tetrahymena, the engulfed digoxin appeared in the epiplasmic layer and also in the cilia after digitoxin pretreatment. Digoxin pretreatment did not increase digoxin uptake. These data indicate that Tetrahymena has: (1) the capacity to discriminate between closely related molecules; (2) the ability to induce digoxin-like material production; and/or (3) enzymes that can effect a digitoxin-digoxin transformation.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis

Earlier experiments demonstrated the presence of hormones of the higher ranked animals in Tetrahymena. In the present experiments two fixatives, paraformaldehyde, which is commonly used and a carbodiimide, EDAC that was recommended by Panula et al. for the immunocytochemistry of histamine, are compared in Tetrahymena for the demonstration of histamine and serotonin by using flow cytometry and confocal microscopy. Both hormone levels were significantly higher after EDAC fixation; serotonin almost doubled and histamine was more than fivefold. The confocal microscopic pictures were clearer and the hormones' localization was easier. The results support earlier observations on the presence of these hormones in Tetrahymena and points to the advantage of EDAC fixation for demonstrating these hormones immunocytochemically.     

Behavior of the Contractile Vacuole of Tetrahymena pyriformis W: A Redescription with Comments on the Terminology

SYNOPSIS. The behavior of the contractile vacuole of Tetrahymena pyriformis W has been recorded and analyzed quantitatively by cinephotography. The vacuole fills in a stepwise fashion by the confluence of ampullae which appear regularly at the beginning of systole and whose membranes are continuous with that of the contractile vacuole throughout the cycle. The vacuole may subsequently fill slowly by a means not discernible by light microscopy. The vacuole rounds up at the beginning of systole and shortly thereafter the ampullae reappear around the perimeter of the vacuole. They are expanded by fluid forced into them from the vacuole. Round-up and the mode of growth of the ampullae indicate that the contractile vacuole is truly contractile. Expulsion occurs soon after the appearance of the ampullae and terminates the cycle. Contraction is initiated at regular intervals by a timing mechanism which is independent of the size of the vacuole. Suitable terminology to describe the structure and behavior of the contractile vacuole is discussed.     

Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition

ABSTRACT We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca²⁺ (68 μM) and Mg²⁺ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca²⁺ and Mg²⁺ can be abolished by the addition of insulin (10^-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 10⁴ cell·ml⁻¹ or more, the effect of shaking was absent. However, with inoculates of 10³ or 10² cell·ml⁻¹, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.     

Evolutionary adaptation to environmental stressors: a common response at the proteomic level

Mechanistic trade‐offs between traits under selection can shape and constrain evolutionary adaptation to environmental stressors. However, our knowledge of the quantitative and qualitative overlap in the molecular machinery among stress tolerance traits is highly restricted by the challenges of comparing and interpreting data between separate studies and laboratories, as well as to extrapolating between different levels of biological organization. We investigated the expression of the constitutive proteome (833 proteins) of 35 Drosophila melanogaster replicate populations artificially selected for increased resistance to six different environmental stressors. The evolved proteomes were significantly differentiated from replicated control lines. A targeted analysis of the constitutive proteomes revealed a regime‐specific selection response among heat‐shock proteins, which provides evidence that selection also adjusts the constitutive expression of these molecular chaperones. Although the selection response in some proteins was regime specific, the results were dominated by evidence for a “common stress response.” With the exception of high temperature survival, we found no evidence for negative correlations between environmental stress resistance traits, meaning that evolutionary adaptation is not constrained by mechanistic trade‐offs in regulation of functional important proteins. Instead, standing genetic variation and genetic trade‐offs outside regulatory domains likely constrain the evolutionary responses in natural populations.     

Chemotactic activity of oligopeptides containing an EWS motif on Tetrahymena pyriformis: the effect of amidation of the C-terminal residue

Chemotactic properties of 3-7-mer peptides containing an EWS motive and their peptide amides synthesized and characterized by us were investigated in Tetrahymena pyriformis GL model. Analysis of the peptide acids shows that SEWS possesses exceptionally strong (660%+/-21; 430%+/-18) chemoattractant ability at 10(-12) and 10(-11) m respectively. The shorter peptide (EWS) possesses chemorepellent activity, while longer peptides display neutral (WSEWS) or moderate chemoattractant (EWSEWS and GEWSEWS) chemotactic ability. Amidation of the C-terminus can significantly modify the character of peptides: it points to the conclusion that a free alpha-COOH group at this position is required for the high efficiency of SEWS, while in the shorter (EWS) and longer peptides (WSEWS and EWSEWS) amidation can result in chemoattractant ligands. Evaluation of the structure-function relationship of these compounds establishes the significance of Glu (E) with its high surface-exposed area and negatively-charged side chain. The high discriminative ability and good chemotactic responsiveness of Tetrahymena support the theory that a chemotactic signalling mechanism working in higher levels of phylogeny is a well conserved and inducible one even in protozoa.