Effects of indomethacin on the divisional morphogenesis and cytoskeleton-dependent processes of Tetrahymena

Indomethacin (0.1 mM) causes significantly altered phospholipid synthesis in Tetrahymena and is able to influence the inositol phospholipid signalling system (9). In the present study the effects of indomethacin on the course of cell division, cyclin expression, the cortical microtubular system and on cytoskeleton-dependent processes (motility, phagocytosis) were investigated. As expected from its interference with the synthesis of phospholipids, indomethacin affected Tetrahymena in a number of ways: the structure of the cortical microtubular system became irregular; in many cells the stomatogenesis (development of new oral apparatus) and the development of the fission furrow was not accompanied by elongation of the macronucleus, which is a typical phenomenon of the normal course of mitosis: apparently indomethacin uncouples these phenomena. After indomethacin treatment, the expression of both cyclin A and cyclin B(1) were reduced significantly. The cell growth rate, motility and phagocytotic activity were all considerably reduced. There are probably additional mechanisms responsible for the effect of indomethacin on the systems that control divisional morphogenesis, for microtubule-dependent processes and for the connection between nuclear and cortical alterations during the cell cycle. Effects on protein phosphorylation/dephosphorylation, on cyclin expression and on microtubular functions are probably involved. These possibilities are discussed.     

Effect of drugs affecting microtubular assembly on microtubules, phospholipid synthesis and physiological indices (signalling, growth, motility and phagocytosis) in Tetrahymena pyriformis

Structural changes of microtubules, incorporation of radioactively labelled components into phospholipids, cell motility, growth and phagocytosis were studied under the effect of four drugs affecting microtubular assembly: colchicine, nocodazole, vinblastine and taxol. Although the first three agents influence microtubules in the direction of depolymerization and the fourth stabilizes them, their effects on the structure of microtubules cannot be explained by this. Using confocal microscopy after an acetylated anti-tubulin label, in nocodazole- and colchicine-treated cells, the basal body cages disappear and longitudinal microtubules (LM) became thinner without changing transversal microtubules (TM). After taxol treatment LM also became thinner, however TM disappeared. Under the effect of vinblastine TM became thinner, without influencing LM. These drugs influence the incorporation of components ([(3)H]-serine, [(3)H]-palmitic acid and (32)P) into phospholipids, however their effect is equivocal and cannot be consequently coupled with the effect on the microtubules. Nocodazole, vinblastine and taxol significantly reduced the cell's motility, however colchicine did so to a lesser degree. Vinblastine and nocodazole totally inhibited, and taxol significantly decreased cell growth, while colchicine in a lower concentration increased the multiplication of cells. Phagocytosis was not significantly influenced after 1 min, but after 5 min all the agents studied (except colchicine) significantly inhibited phagocytosis. After 15 and 30 min each molecule caused highly significant inhibition. The experiments demonstrate that drugs affecting microtubular assembly dynamics influence differently the diverse (longitudinal, transversal etc.) microtubular systems of Tetrahymena and also differently influence microtubule-dependent physiological processes. The latter are more dependent on microtubular dynamics than are changes in phospholipid signalling.     

Effects of sodium fluoride (NaF) on the cilia and microtubular system of Tetrahymena

The effect of the nucleophilic reagent NaF on the microtubular system of Tetrahymena was studied by using scanning electron microscopy (SEM), confocal microscopy, and flow cytometry. Treatments with 40 mM NaF significantly reduced the amount of alpha-tubulin while 80 mM treatment did not alter its quantity. One possible explanation for this alpha-tubulin overexpression is that the higher amount of alpha-tubulin enables this organism to carry out the appropriate function of the cytoskeleton under this undesirable influence of higher amounts of 80 nM NaF. However, the amount of acetylated tubulin increased in a dose-dependent manner. The cilia became fragile under the effect of 80 mM NaF. Confocal microscopy revealed that after 40 mM NaF treatment transversal microtubule bands (TMs) and longitudinal microtubule bands (LMs) as well as basal bodies (BBs) were extremely strong decorated with anti-acetylated tubulin antibody and TM-localization abnormalities were visible. In the 80 mM NaF-treated cells, the deep fiber of oral apparatus was very strongly labeled, while the TMs and LMs were less decorated with anti-acetylated tubulin antibody, and LM deformities were visible. It is supposed that post-translational tubulin modifications (e.g., acetylation) defend the microtubules against the NaF-induced injury. NaF is able to influence the activity of several enzymes and G-proteins, therefore is capable to alter the structure, metabolism, and the dynamics of microtubular system. The possible connection of signaling and cytoskeletal system in Tetrahymena is discussed.     

Effects of choline and ethanolamine on the synthesis and breakdown of the inositol phospholipid (PI) system in Tetrahymena

Lower concentrations of choline chloride and ethanolamine (10^?3 M ; 10^?5 M ) increased phosphatidyl inositol (PI), phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bisphosphate (PIP₂) level of Tetrahymena, while higher concentrations (10^?2 M ) decreased them. These two substances also influenced, however in a less obvious way, the transformation of inositol phospholipids. The experiments draw attention to the sensitivity of the precursors of the second messenger system at a phylogenetically low level.     

RanGTPase激活蛋白RanGAP在嗜热四膜虫细胞中的定位及功能

RanGTPase激活蛋白（RanGTPase activating protein,RanGAP）和Ran相互作用,提高了Ran GTPase水解GTP的效率. RanGAP参与细胞内核质运输、纺锤体组装、核膜重建和异染色质的组装．生物进化过程中,不同生物的RanGAP表现出结构和功能的多样性．本研究从嗜热四膜虫大核基因组中鉴定出1个保守的RanGTPase激活蛋白基因RanGAP（TTHERM_00766430）．实时荧光定量PCR表明,RanGAP在四膜虫营养生长、饥饿和有性生殖过程中均有表达,且在有性生殖4～6 h表达水平最高．免疫荧光定位表明,在营养生长期、饥饿期及有性生殖的早期,RanGAP定位于细胞质中| 在有性生殖后期, RanGAP定位于凋亡的大核中．过表达RanGAP的细胞增殖速率下降,大核分裂和胞质缢缩异常, 产生无大核细胞．敲减RanGAP的细胞大核形态异常,细胞增殖速率下降,无丝分裂受到抑制,进而产生无大核细胞．RanGAP的过表达或敲除分别引起四膜虫RAN1,RanBP1和RCC1基因的表达下调或上调．结果表明,RanGAP通过Ran信号通路调控了嗜热四膜虫无性生殖过程中大核的无丝分裂,并可能参与了有性生殖过程中亲本大核的凋亡．     

Effects of synthetic peptides on thylakoid phosphoprotein phosphatase reactions

A synthetic peptide analogue of the phosphorylation site of LHC II, when phosphorylated by thylakoid membranes, served as a substrate for the thylakoid phosphoprotein phosphatase. The phosphopeptide became dephosphorylated at a low rate, comparable to that of the 9 kDa phosphoprotein. Phospho-LHC II itself became dephosphorylated much more rapidly, at a rate unaffected by endogenous phosphorylation of the peptide. Endogenous phosphorylation of the peptide was also without effect on other thylakoid protein phosphorylation and dephosphorylation reactions. In contrast, dephosphorylation of many thylakoid phosphoproteins was inhibited by addition of a pure, chemically-synthesised phosphopeptide analogue of phospho-LHC II. This result suggests that at least one thylakoid phosphoprotein phosphatase exhibits a broad substrate specificity. The results indicate that any one of a number of amino acid sequences can give a phosphoprotein configuration that is recognised by a single phosphatase.     

Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions

Phosphatidylinositol (PtdIns) synthase in microsomal fractions derived from Tetrahymena vorax was studied to determine its activity requirements. The suitability of inositol isomers as substrates for the synthase and in headgroup exchange reactions also was investigated. Tetrahymena PtdIn synthase activity was optimum in the presence of 2 mM MgCl2 plus 2 mM MnCl2, a pH of 7.8, and a temperature of 30 degrees C. The enzyme retained approximately 80% of its activity after incubation at 70 degrees C for 10 min. PtdIns headgroup exchange activity was maximal in the presence of cytidine monophosphate. By following either the accumulation of radiolabeled reaction products or the loss of radiolabel from precursors, each of the inositol isomers tested appeared to serve as substrates for both the PtdIns synthase and PtdIns:inositol phosphatidyl transferase activities. In each case, myo-inositol and scyllo-inositol were the preferred substrates. The data suggest two routes for the formation of phosphatidyl-non-myo-inositols in Tetrahymena and the potential for the production of novel, non-myo-inositol-containing second messengers.     

Tetrahymena Tubulins and In Vitro Translation of Tetrahymena RNA*

SYNOPSIS. Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymenaα tubulin did not comigrate with either brain or flagellar α tubulins, although brain, flagellar, and ciliary β tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W. S. HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A + RNA had 2 prominent protein bands which comigrated with α and β tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products ofpoly-A- RNA.     

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.     

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.     

Effects of taxol treatment on the microtubular system and mitochondria of Tetrahymena

Complex investigation was done using immunocytochemical confocal microscopy, electron microscopy and flow cytometry on the effect of taxol to the microtubular arrangement and dynamics. The most interesting phenomenon was the rapid disappearance of transversal microtubule bands, while longitudinal microtubule bands remained and were submitted to the known effects of taxol. There was a broad variation in mitochondrial effect, some of them remained normal, while others swollen, desintegrated and their tubules disoriented. Treatment with 50 nM taxol significantly reduced the binding of anti alpha-tubulin antibody and a lesser degree anti-acetylated tubulin antibody. The difference between the transversal and longitudinal microtubules is emphasized by the results and the paper discusses the possibilities of indirect effects of taxol to the transversal microtubules (tubulin-GTP interaction, faster turnover, mitochondrial interaction). Polyglutamylation of tubulin has not a role in this difference.     

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.     

pH-Dependent Effects of 2,4-Dinitrophenol (DNP) on Proliferation, Endocytosis, Fine Structure and DNP Resistance in Tetrahymena

ABSTRACT. In view of the importance of external pH on cytotoxic effects of ionizable agents, the pH-dependent effects of 2,4-dinitrophenol (DNP) were investigated. As uncoupler of oxidative phosphorylation. DNP interferes with the proton gradient across the inner mitochondrial membrane. DNP was added to proliferating Tetrahymena pyriformis in media of different initial pH. Effects studied were rates of cell proliferation and endocytosis, and fine structure. Findings correlated with the calculated concentration of undissociated DNP, taking into account that pH changes with time and cell density in Tetrahymena cultures. A linear relationship thus emerged between initial concentrations of undissociated DNP and lengths of the lag preceding cell proliferation. Once resumed, the rate of proliferation corresponded to that of control cells, even in different concentrations of undissociated DNP, presumably indicating an adaptation mechanism. Endocytosis was elevated throughout a wide range of undissociated DNP concentrations with a sharp transition towards inhibition at high DNP concentrations causing lethality with time. Changes in fine structure of DNP-treated cells (mitochondria, peroxisomes, nucleoli) also depended on the concentration of undissociated DNP.     

Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide

Abscisic acid-induced gene-expression requires the activity of protein(s) sensitive to the protein-tyrosine phosphatase inhibitor phenylarsine oxide. It is generally accepted that phosphorylation/dephosphorylation of proteins plays an important role in signal transduction cascades. evidence is now accumulating that for plants the same holds true. To study the role of phosphorylation in ABA signal transduction, we used six different compounds which were reported to inhibit phosphatase action. Three of these inhibitors: phenylarsine oxide (PAO), Calyculin A (CA) and Okadaic Acid (OA) appeared capable of inhibiting ABA-induced gene-expression. The same three inhibitors are shown to bring about hyperphosphorylation of two approximately 40 kDa proteins, present in the membrane-bound fraction of barley aleurone cells. The other three inhibitors had no visible effect on the phosphorylation status of the barley proteins. The hyperphosphorylation of the two 40 kDa proteins coincided with an increase of tyrosine-phosphorylation of two 40 kDa proteins with different pI, as determined with anti-phosphotyrosine antibodies.     

Effects of cytochrome P450 inhibitors on agonist-induced Ca2+ responses and production of NO and PGI2 in vascular endothelial cells

The gap junction protein connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).     

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.     

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.     

Effects of polyamine hydrochlorides and salts on phosphoprotein phosphatase.

Polyamine hydrochlorides, NaCl and magnesium acetate stimulated the enzymatic dephosphorylation of phosphorylated H2B histone by two forms (large form, mol. wt. 250 000; small form, mol. wt. 30 000) of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16). These ionic compounds stimulated the large form of the enzyme 5--9-fold but stimulated the small form of theenzyme only 2-fold. With phosphorylated H2B histone as substrate, these effectors caused an increase in both Km and V values of the two forms of the enzyme. On the other hand, when a tryptic phosphodecapeptide derived from phosphorylated H2B histone was used as substrate, these effectors were always inhibitory apparently non-competitively with respect to the substrate. Using phosphorylated H1 histone as substrate, these effectors stimulated the large form of the enzyme 2-fold but inhibited the small form. With phosphorylase a as substrate, the reactions were also inhibited by these effectors irrespective of the enzyme employed. With respect to phosphorylase a, this inhibition was apparently of a competitive type for the large form and a non-competitive type for the small form of the enzyme.