Tetrahymena Pyriformis GL Research Articles

Comparisons among six strains of Tetrahymena by microcalorimetry

The typical power-time curves of six strains of Tetrahymena were determined by microcalorimetry at 28 A degrees C. Metabolic characteristics were obtained including metabolic rate constant (r), heat output in the log phase (Q (log)), total heat production (Q (T)), peak time, and measured and potential maximum power output (P (m) and P (max)). The power-time curves and metabolic characteristics show the differences among Tetrahymena strains. Euclidean distances and a clustering analysis diagram were obtained by metabolic properties. The smaller distances are between the four strains of Tetrahymena thermophila and the largest distance exists between Tetrahymena pyriformis GL and Tetrahymena pigmentosa HG2. These results are consistent with those obtained by first internal transcriptional spacer sequences. Therefore, the power-time curves and metabolic properties can be noticeable characteristics in distinguishing the different strains of Tetrahymena. And, the heat output in the log phase (Q (log)) is probably the most specific characteristic in distinguishing the different Tetrahymena strains and studying their relationships.

Studies on the nongrowth metabolism of the different strains of Tetrahymena cells by isothermal microcalorimetry

The nongrowth metabolic processes of Tetrahymena strains with the different cell densities were monitored by isothermal microcalorimetry, including Tetrahymena thermophila BF1, Tetrahymena thermophila SB210, Tetrahymena pyriformis GL, the mixed cells of T. thermophila BF1 and T. thermophila SB210, and the mixed cells of T. thermophila BF1 and T. pyriformis GL. All the typical power-time curves showed a decreasing trend on the whole. It was found that total heat production (Q (t)) and maximum heat output (P (m)) decreased significantly with the decrease of cell density. Cell density did not influence significantly the maximum heat output per cell (P (cell)) of Tetrahymena cells, which was probably due to enough oxygen. The P (cell) and metabolic decrease rate constant (K) values for the mixture of T. thermophila BF1 and T. pyriformis GL, and that of T. thermophila BF1 and T. thermophila SB210 were similar to the mean values of P (cell) and K for the corresponding single Tetrahymena strain, respectively. It was speculated that the cell mixing did not possibly influence significantly their P (cell) and K values because of no competition for nutrition, oxygen, and room, and the low conjunction percentages.

Characterization of Deciliation-Regeneration Process of Tetrahymena Pyriformis for Cellular Robot Fabrication

Artificial magnetotactic Tetrahymena pyriformis GL ( T. pyriformis) cells were created by the internalization of iron oxide nano particles and became controllable with a time-varying external magnetic field. Thus, T. pyriformis can be utilized as a cellular robot to conduct micro-scale tasks such as transportation and manipulation. To complete these tasks, loading inorganic or organic materials onto the cell body is essential, but functionalization of the cell membrane is obstructed by their motile organelles, cilia. Dibucaine HCl, a local anesthetic, removes the cilia from the cell body, and the functional group would be absorbed more efficiently during cilia regeneration. In this paper, we characterize the recovery of artificial magnetotactic T. pyriformis after the deciliation process to optimize a cellular robot fabrication process. After sufficient time to recover, the motility rate and the average velocity of the deciliated cells were six and ten percent lower than that of non-deciliated cells, respectively. We showed that the motile cells after recovery can still be controlled using magnetotaxis, making T. pyriformis a good candidate to be used as a cellular robot.

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 degrees 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.

Features of mitochondrial energetics in living unicellular eukaryote Tetrahymena pyriformis. A model for study of mammalian intracellular adaptation

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as Delta psi (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP(4-) and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low Delta psi value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the Delta psi-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.

Comparison of the binding of anti-tubulin antibody and the fluorescent taxol derivative Flutax-1 to the microtubular system of Tetrahymena

Using confocal microscopic analysis, FITC-labelled anti-alpha-tubulin antibody and the fluorescent taxol derivative Flutax-1 in fixed and living Tetrahymena pyriformis GL, longitudinal microtubules, oral and somatic cilia, deep fibers, and contractile vacuole pores were equally labeled. While the antibody stained transversal microtubules, these were not labeled by Flutax-1. At the same time, oral cilia were more intensely stained by Flutax-1, than by the antibody. There were no differences in the staining of fixed preparations and living cells. The observations suggest (i) the difference between the MAPs of longitudinal and transversal microtubules which allow or inhibit the binding of the indicator molecules, and (ii) the different functions of these two types of microtubules.

Effect of starvation on insulin production and insulin binding in Tetrahymena

Tetrahymena pyriformis GL was starved for 24 h and then the immunologically demonstrable insulin content and FITC-insulin binding were measured by flow cytometry and localization was studied by confocal microscopy. The amount of endogeneous insulin as well as FITC insulin binding, was highly significantly elevated. Glucose feeding for 30 min abolished the elevation of FITC-insulin binding. In starved cells, insulin-binding sites disappeared from the surface and FITC-insulin was bound inside the cells, within large food vacuoles. Endogeneous insulin was dispersed in the cytoplasm both in the control and starved cells and food vacuoles did not contain it. The results call attention to the stimulatory effect of starvation on insulin production in Tetrahymena, in parallel with the internal storage of insulin receptors, which points to an autocrine mechanism.

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis

The chemotactic properties of tuftsin (H-TKPR-OH), tuftsin derivatives (H-KPR-OH, H-TKPKG-NH(2), Ac-TKPKG-NH(2)) and TKPKG-based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N-terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C-terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10(-9)-10(-6) M), while T30 had a wide-ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin-induced chemotaxis results from fairly short-term signalling in Tetrahymena.

Effect of the Polylysine Based Polymeric Polypeptides on the Growth and Chemotaxis of Tetrahymena Pyriformis

Polylysine based branched polypeptides represents a group of biocompatible polymers that could be utilized as macromolecular carriers for drugs, epitopes or reporter molecules. Ten polymers with different character (amino acid composition and charge properties) were prepared: polypeptides with single amino acid in the branches (poly[Lys(Xi)]), X = His, Pro or Glu; and polymers possessing oligo[DL-alanine] side chains only (poly[Lys(DL-Alam) (AK) or with an additional amino acid residue poly[Lys(Xi-DL-Alam)] (XAK), where X = Ser (SAK), Thr (TAK), Glu (EAK), acetyl-Glu (Ac-EAK) or succinyl-Glu (Succ-EAK). were investigated. The concentration of these compounds influence the chemotaxis and survival of eukaryotic unicellular model organism, Tetrahymena pyriformis GL. Two types of experiments were performed. First the polymer induced chemoattractant/chemorepellent response of Tetrahymena cells were tested, then chemotactic selection experiments were performed. The chemotactic responses elicited by the polymers were dependent not only on chemical properties (composition, charge and the length of the side chain) of the compounds, but also on their concentration. Based on these results, the polymers were grouped as full-chemoattractant expressing this behavior in the full concentration range investigated (HiK), full-chemorepellent (EiK and Ac-EAK) and partial chemoattractant/chemorepellent with concentration dependent activity (PiK, EAK and Succ-EAK).

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.

Chemotactic selection with insulin, di-iodotyrosine and histamine alters the phagocytotic responsiveness of Tetrahymena

Chemotactic selection is a method by which populations of cells exposed to ligands can be isolated and subsequently cultivated. We used Tetrahymena pyriformis GL cultures selected by chemotactic selection to insulin (10 nM), histamine (0.1 nM) and di-iodotyrosine (T2, 10 nM) to study the phagocytotic capacity under the induction of selector hormones. Our results show a long-lasting link between chemotactically selected cultures and phagocytotic activity. Cells selected to histamine produced the highest phagocytotic activity upon a second exposure to the selector hormone. T2 selection was also strongly effective, however, the phagocytosis stimulation was not specific to the hormone given later. Insulin selected sub-populations had different phagocytotic responses to the control substance itself, whereas histamine selected sub-populations seem to be heterogeneous in the phagocytotic response to histamine. For insulin, the increased endocytotic or metabolic activity was demonstrated by the lack of non-phagocytotic cells. These experiments call attention to the evolutionary role of selection in the later developing receptor-hormone relationship.

On macronuclear “subnuclei”, or genome segregation, and “amitosis” in amicronucleate Tetrahymena pyriformis GL. A study utilizing the effect of vanadate on nuclear division

Summary In 1940, Tetrahymena pyriformis GL was reported amicronucleate by W. H. Furgason. Unable to renew its genetic complement through conjugation, the ciliate still thrives with unchanged phenotype. This fact indicates physical integrity of the haploid macronuclear genome (5 “chromosomes”) during cell division, as also reflected in 90 unitsized chromatin aggregates (“subnuclei”) distributed equally to the daughters. This stands in contrast to biochemical sizing of bulk macronuclear DNA in thousands of subchromosomal DNA fragments distributed randomly during cell division. The purpose was to investigate the problem utilizing vanadate that affects nuclear division so that the daughters may receive anything from the entire G2-nucleus to no nucleus at all [39]; in theory a single aggregate suffices to survive. Single cell cultures of daughters, derived from cells divided in vanadate, revealed survival of all, but not anucleate, daughters and progeny of unchanged phenotype, hence indicating that the macronuclear chromatin aggregates contain full genetic complement. “Amitosis” in Tetrahymena is genome segregation but equal karyokinesis depends on correct positioning of the macronucleus, properly related to its anterior and posterior movements prior to elongation as though it becomes anchored to both cell poles. Vanadate disturbed correct positioning of the nucleus.

Toxicity of the herbicide chlorpropham towards the freshwater ciliated protozoan Tetrahymena pyriformis GL

Summary Chlorpropham (CIPC), widely used in agriculture as a herbicide, displays genotoxic and antimitotic properties. Its toxic action was studied on the ubiquitous freshwater ciliate Tetrahymena pyriformis, in short term (six hours) and longer term (24 hours) experiments. The results show that CIPC induces an inhibition of cell division during the first hours of treatment. A slow recovery of cell multiplication was then observed. CIPC treatment in both short and long-term experiments affected the somatic ciliature pattern equally, with irregular arrangement of kineties, giving daughter cells exhibiting an aberrant distribution of nuclear apparatus. Specific toxic action of CIPC was detected on the macronuclear structure, inducing a segregation and concentration of nucleoli. DNA and RNA synthesis was clearly disturbed in the presence of CIPC, DNA synthesis decreasing by about 20% during the four first hours of treatment, and RNA synthesis by about 50% after 24 hours. Hence CIPC toxicity is lasting, still disturbing cells even after several generations.

Effects of chelators on the acute toxicity and bioavailability of aluminium to Tetrahymena pyriformis

The acute toxic effects of aluminium (Al) to the ciliated protozoa Tetrahymena pyriformis GL (TP) were investigated after a short exposure of 9 h, a time which normally allowed three generations of T. pyriformis in control culture to be obtained. Al was administered under the soluble forms of three salts (AlCl 3, Al(NO 3) 3, Al 2(SO 4) 3) and the fine particles of an insoluble salt (Al 2O 3), with or without chelators (humic acids, citric acid, EDTA). A significant and a dose-dependent inhibition of the growth rate and of the motility was observed with TP exposed to Al compounds. The IC 50 values of Al were ranked between 10 and 15 μg Al/ml, whatever the soluble Al salt may be; it was increased to 495 μg Al/ml with Al 2O 3. The Al chelates of citrate and EDTA did not significantly modify the toxicity of Al to TP. Furthermore, an increased and unexpected toxicity of Al was observed with Al humate chelates. This phenomena can be explained by the stimulation of the phagocytosis rate of TP by the particles of organic humic matter, which consequently induced a rapid increase of the amount of Al ingested by TP. Moreover, these toxicological results were confirmed by the intracellular quantification of Al by atomic absorption spectrometry and by the localization of Al in TP by an ultrastructural analysis, performed with an electron microscopy and a X-ray probe.

The Effect of β-Chloro D-Alanine and L-Cycloserine on The Serine, Phosphorus and Palmitic Acid Uptake and Metabolism of Tetrahymena Lipids

The serine palmitoyltransferase inhibitors beta-chloro-D-alanine and L-cycloserine resulted in the uptake and metabolism of 3H-serine, 3H-palmitic acid and 32P significant alterations in the unicellular Tetrahymena pyriformis GL as compared to the untreated cells. In contrast with the higher eukariotic cells, by these treatments - except 5 mM L-cycloserine - the ceramide formation were not inhibited in Tetrahymena. L-cycloserine inhibited the conversion of phosphatidylserine (PS) to phosphatidyl-ethanolamine (PE) by decarboxylation, and the conversion of PE to phosphatidylcoline (PC) by methylation. The shorter L-cycloserine treatments caused lower, and the longer treatments higher label in glycerophospholipids. beta-chloro-D-alanine resulted in the glycerophospholids higher lipid precursor incorporation both in the shorter and longer treatments. Presumably beta-chloro-D-alanine treatments inhibit the transaminase activity, and the higher concentration (5 and 10 mM) proved to be toxic for Tetrahymena. We found differences between the metabolism of serine and palmitic acid labeled lipids in the beta-chloro-D-alanine and L-cycloserine treated groups. This phenomenon is probably due to a difference in the uptake of phospholipid head group component serine and hydrophobic tail precursor palmitic acid: the incorporation of palmitic acid in Tetrahymena is extremely quick, on the other hand, the uptake of serine is slower, a clear time dependence was measured.

LOCALIZATION OF β-ENDORPHIN IN TETRAHYMENA BY CONFOCAL MICROSCOPY. INDUCTION OF THE PROLONGED PRODUCTION OF THE HORMONE BY HORMONAL IMPRINTING

The unicellular Tetrahymena has hormone receptors and hormones which are characteristic of higher vertebrates, as well as similar signal transduction pathways. In the present experiments, immunocytochemistry and confocal microscopy were used to study the presence and localization of β-endorphin in Tetrahymena pyriformis GL. Endorphin (or endorphin-like material) was localized in the cortical structures, oral field, cilia and nuclear envelope. One-hour treatment with β-endorphin (‘hormonal imprinting’) increased the presence of immunocytochemically demonstrable endorphin immediately and after 24h, and was especially strong after 96h of treatment. Simultaneous treatment with naloxone, an opioid antagonist, did not inhibit endorphin effect, but had an additive effect on endorphin production. Naloxone alone induced a very intensive accumulation of endorphin 96h after treatment. The results support the possibility of a hormone production being induced by the imprinting procedure, but the imprinter-like effect of naloxone also points to the importance in this case of non-discriminatory receptors also being involved in the process.

The histidine decarboxylase (HDC) gene of Tetrahymena pyriformis is similar to the mammalian one. A study of HDC expression.

RNA was isolated from Tetrahymena pyriformis GL and using human histidine decarboxylase (HDC) gene primers, the RT-PCR product was sequenced. A fraction containing 207 base pairs was compared to the published sequences of prokaryotic and mammalian (rat, mouse and human) HDC cDNA (exons). The HDC-cDNA fraction of Tetrahymena was similar to the mammalian cDNA-s and it was completely different from the prokaryotic HDC-gene. The results indicate the presence of a mammalian-like HDC-gene already in a unicellular eukaryote organism and demonstrates also that the divergence of the prokaryotic-eukaryotic common gene took place already at this low evolutionary level.

PLA 2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A 2 (PLA 2) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA 2-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca 2+-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA 2 activity and the release of free arachidonic acid (AA) from 1-acyl 2- 14C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA 2 hyperactivity is Ca 2+-dependent. There was no considerable alteration in the PLA 2 activity by stimulation of the activity by tyrosine kinase (with vanadate, H 20 2), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF 4 thus in Tetrahymena PLA 2 activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA 2 activity is in connection with the phospholipid metabolism of Tetrahymena.

Phospholipase D activity in the Tetrahymena pyriformis GL.

Phospholipase D (PLD) is an enzyme which participates in the signaling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signaling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena.

Protein Prenylation in Tetrahymena

Summary Tetrahymena vorax V 2S and Tetrahymena pyriformis GL were examined for the presence of proteins covalently modified with mevalonate-derived lipids. Radiolabeling of cells using [3H]mevalonolactone, [ 3 H]mevalonate, [ 3 H]farnesyl pyrophosphate or [ 3 H]geranylgeranyl pyrophosphate resulted in the appearance of protein-associated radioactivity following protein delipidation in acetone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled protein revealed radioactive polypeptides ranging in molecular mass from 123 to 14 kDa following scintillation counting of gel slices or fluorography. Methyl iodide cleavage of total labeled protein followed by HPLC analysis and scintillation counting of the released prenyl alcohols showed radioactive peaks that co-chromatographed with commercially obtained farnesol (C 15 ) and geranylgeraniol (C 20 ).