Stereochemical investigations of the Tetrahymena cyclase, a model system for euphane/tirucallane biosynthesis.

The squalene cyclase of Tetrahymena pyriformis cyclizes 2,3-dihydrosqualene to euph-7-ene. This was used as a model for euphane biosynthesis. D-ring formation was demonstrated to involve pre-chair folding through an experiment with 2,3-dihydro-5-oxasqualene. This requires that a non-least motion rotation (120°) of the C17-20 bond in the intermediate deoxydammaranyl cation occurs before the first 1,2-hydride shift. Truncated substrates relieved the hindrance associated with this rotation and permitted a least motion pathway. Several triterpenes were found to be minor products of the Tetrahymena cyclase.

Evidence for N-Terminal Myristoylation of Tetrahymena Arginine Kinase Using Peptide Mass Fingerprinting Analysis.

In this study, we confirmed N-terminal myristoylation of Tetrahymena pyriformis arginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H(+)) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesized T. pyriformis AK1 is myristoylated. The fixed specimens of T. pyriformis were reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate that T. pyriformis AK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.

Cooperativity and evolution of Tetrahymena two-domain arginine kinase.

Tetrahymena pyriformis contains two arginine kinases, a 40-kDa enzyme (AK1) with a myristoylation signal sequence at the N-terminus and a two-domain 80-kDa enzyme (AK2). The former is localized mainly in cilia and the latter is in the cytoplasm. AK1 was successfully synthesized using an insect cell-free protein synthesis system and subjected to peptide mass fingerprinting (PMF) analysis. The masses corresponding to unmodified N-terminal tryptic peptide or N-terminal myristoylated peptide were not observed, suggesting that N-terminal peptides were not ionized in this analysis. We performed PMF analyses for two other phosphagen kinases (PKs) with myristoylation signals, an AK from Nematostella vectensis and a PK from Ectocarpus siliculosus. In both cases, the myristoylated, N-terminal peptides were clearly identified. The differences between the experimental and theoretical masses were within 0.0165-0.0583 Da, supporting the accuracy of the identification. Domains 1 and 2 of Tetrahymena two-domain AK2 were expressed separately in Escherichia coli and the extent of cooperativity was estimated on the basis of their kinetic constants. The results suggested that each of the domains functions independently, namely no cooperativity is displayed between the two domains. This is in sharp contrast to the two-domain AK from Anthopleura.

Two arginine kinases of Tetrahymena pyriformis: characterization and localization.

Two cDNAs, one coding a typical 40-kDa arginine kinase (AK1) and the other coding a two-domain 80-kDa enzyme (AK2), were isolated from ciliate Tetrahymena pyriformis, and their recombinant enzymes were successfully expressed in Escherichia coli. Both enzymes had an activity comparable to those of typical invertebrate AKs. Interestingly, the amino acid sequence of T. pyriformis AK1, but not AK2, had a distinct myristoylation signal sequence at the N-terminus, suggesting that 40-kDa AK1 targets the membrane. Moreover, Western blot analysis showed that the AK1 is mainly localized in the ciliary fraction. Based on these results, we discuss the phosphoarginine shuttle, which enables a continuous energy flow to dynein for ciliary movement in T. pyriformis, and the role of AK1 in this model.

Identification and characterization of the arsenite methyltransferase from a protozoan, Tetrahymena pyriformis.

Arsenic (As) methylation in aquatic microbes plays a major role in the biogeochemistry of As. Protozoa, especially the free-living freshwater species, are important players in aquatic ecological health. In this study, an arsenite (As(III)) methyltransferase, TpyArsM, was identified and characterized in a free-living protozoan, Tetrahymena pyriformis. In order to confirm its function, TpyarsM gene was knocked-out in Tetrahymena and was also heterologously expressed in hypersensitive E. coli; these events resulted in expected decreases in As tolerance and methylation ability, respectively. In-vitro tests revealed that purified TpyArsM protein methylated inorganic As to mono- and di- methylarsenate, and also had the novel property of producing trimethylarsenite (TMA(III)) and dimethylarsine (Me2AsH) gases. This new methyltransferase gene, identified in a species near the base of the food web, has enriched our knowledge of As methyltransferases and has great potential for bioremediation of As-contaminated environments.

[Effect of natural amino acids and sugars on cyclase activities in infusoria Tetrahymena pyriformis and Dileptus anser].

Natural amino acids and sugars in intracellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, thyrosine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis are increased in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large spectrum of comparatively simple natural molecules has a hormone-like action.

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.

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 T(3)). Here, the effect of other stressors (CuSO4 poisoning, tryptophan hydroxylase inhibitor parachlorophenylalanine (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, T(3)) 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.

Toxicity assessment of the herbicides sulcotrione and mesotrione toward two reference environmental microorganisms: Tetrahymena pyriformis and Vibrio fischeri.

The potential toxicity of sulcotrione (2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione) and mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione), two selective triketonic herbicides, was assessed using representative environmental microorganisms frequently used in ecotoxicology: the eukaryote Tetrahymena pyriformis and the prokaryote Vibrio fischeri. The aims were also to evaluate the toxicity of different known degradation products, to compare the toxicity of these herbicides with that of atrazine, and to assess the toxicity of the commercial herbicidal products Mikado and Callisto. Toxicity assays involved the Microtox test, the T. pyriformis population growth impairment test, and the T. pyriformis nonspecific esterase activity test. For each compound, we report original data (IC(50) values) on nontarget cells frequently used in ecotoxicology. Analytical standards sulcotrione and mesotrione showed no toxic effect on T. pyriformis population growth but a toxic influence was observed on nonspecific esterase activities of this microorganism and on metabolism of V. fischeri. Most of the degradation products studied and the two commercial formulations showed a greater toxicity than the parent molecules. Compared with the effect of atrazine, the toxicity of these triketonic herbicides was less than in T. pyriformis and greater than or the same as in V. fischeri. Additional work is needed to obtain a more accurate picture of the environmental impact of these herbicides. It will be necessary in future experiments to study the ecosystemic levels (aquatic and soil compartments) and to assess the potential toxicity of the newly discovered degradation products and of the additives accompanying the active ingredient in the commercial herbicidal formulations.

[Changes in the DNA content in EGF-stimulated ciliate Tetrahymena pyriformis under effect of caffeine, KCl, and inhibitors of PLC and PKC].

It has been established that DNA content in ciliata Tetrahymena pyriformis changes under effect of the caffeine, and PLC and PKC inhibitors (U 73122 and chelerythrine accordingly). 10 mM caffeine stimulated DNA synthesis in these cells, 1 microM U 73122 and 25 microM chelerythrine, on the contrary, blockades the synthesis. 50 and 500 mM KCl, the agent which causes depolarization of plasma membrane stimulating Ca2+ entry into the cells, has no effect. The transduction of the proliferative signals in T. pyriformis cells has been proposed to be of Ca(2+)-dependent nature.

The effect of histone deacetylase inhibitor trichostatin A (TSA) on the incorporation of 32P (Pi) and 3H-palmitic acid into the phospholipids of Tetrahymena.

Histone deacetylases (HDACs) are able to control also the acetylation of tubulin. In the present experiments the effect of trichostatin A (TSA), a HDAC inhibitor was studied on the incorporation of 3H-palmitic acid and 32P to the phospholipids (PI, PIP, PS, PC, PA, PE) of Tetrahymena pyriformis, considering earlier observations on the microtubular system's influence on signalling in this unicellular eukaryote. Treatment with 1, 5, or 10 microM TSA was studied. The incorporation of hydrophobic tail component, palmitic acid was inhibited in a concentration dependent manner into all the phospholipids, except for PA, where the incorporation was increased. 32P incorporation was also inhibited. The possible relation between the microtubular system and signalling is discussed.

Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehyde-3-phosphate dehydrogenase.

Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H(2)O(2) and NO, mainly due to a decrease in its V(max) without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H(2)O(2)-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.

Detection of 1,2-hydride shifts in the formation of euph-7-ene by the squalene-tetrahymanol cyclase of Tetrahymena pyriformis.

Incubation of samples of 2,3-dihydrosqualene, specifically labeled with deuterium at either carbon position 7 or 11, with an enzyme extract from Tetrahymena pyriformis, containing a squalene-tetrahymanol cyclase, provided specimens of euph-7-enes displaying deuterium patterns consistent with the biosynthetic operation of two consecutive 1,2-hydride shifts.

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis.

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.

Enzymatic form and cytoskeletal form of bifunctional Tetrahymena 49kDa protein is regulated by phosphorylation.

Tetrahymena 49kDa protein functions as a citrate synthase (CS) and also assembles to 14-nm filament during cell mating. Bifunctional property of 49kDa protein is suggested to be maintained by the difference of post-translational modification(s). We have found that phosphorylation is present on all three isoforms of 49kDa protein. Dephosphorylation of citrate synthase type isoforms of 49kDa protein, composing pl 7.7 and 8.0 isoforms, reduced its enzymatic activity, shifting these isoforms to basic side. In a course of dephosphorylation, isoform of pl 8.4 appeared with pl 7.7 and 8.0 isoforms, which correspond to the isoforms of 14-nm filament assembling type. With this dephosphorylation, the citrate synthase type isoforms obtained the ability to assemble 14-nm filaments. We propose that enzyme form and cytoskeletal form of 49kDa protein were maintained simply by phosphorylation.

Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization.

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time- and concentration-dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K(m) of 3.7 microM and V(max) 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9-10, was abolished by phenylmethylsulfonyl fluoride and was Ca(2+)- and Mg(2+)-independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.

Use of the ciliated protozoan Tetrahymena pyriformis for the assessment of toxicity and quantitative structure--activity relationships of xenobiotics: comparison with the Microtox test.

Cytotoxicity and quantitative structure-activity relationships of 13 inorganic and 21 organic substances were determined using three bioassays performed on the ciliated protozoan Tetrahymena pyriformis and the luminescent bacterium Vibrio fischeri. The best concordance of toxicity results was observed between the T. pyriformis FDA--esterase activity and population growth inhibition tests for the organic compounds. The sensitivity of these two assays is compared with that of the Microtox test. The T. pyriformis FDA test showed a high sensitivity is most cases. The aim of the current research was to determine whether the relative toxicity of metal ions and organic molecules, with these three bioassays, was predictable using three ion characteristics and hydrophobicity, respectively. For metal ions, the variable that best modeled the toxicity data obtained with the two T. pyriformis tests was the softness index [sigma(p), i.e., (coordinate bond energy of the metal fluoride--coordinate bond energy of the metal iodide)/(coordinate bond energy of the metal fluoride)]. No correlation was found with the Microtox test. For organic compounds, a significant correlation was observed between the hydrophobicity coefficient and the toxicity data. This correlation is closer with the two tests using Tetrahymena.