Effect of difenoconazole fungicide on physiological responses and ultrastructural modifications in model organism Tetrahymena pyriformis.

The continuous and extensive use of pesticides, particularly in the field of agriculture, leads to contamination of all ecosystems (water, soil, and atmosphere). Among pesticides, fungicides constitute a larger group whose impact on the environment are still poorly studied. Difenoconazole belongs to triazole group of fungicides having high photochemical stability and have low biodegradability, which makes them persistent in water bodies. The present study focuses on the physiological and cytotoxic impact of difenoconazole fungicide on ciliated protozoa, Tetrahymena pyriformis with reference to growth, morphology, behaviour and its generation time. Morphological studies showed changes in the shape and size of T. pyriformis. Our result showed an inhibitory effect on population growth of T. pyriformis and the IC50 concentration was found to be 6.8 µg mL-1.The numbers of generations decreased and generation time was found to be extended in a concentration and time dependent manner. Difenoconazole caused significant depletion in phagocytic activity and also ultra-structural changes were observed by Transmission electron microscopy (TEM) analysis. The results indicate that the Tetrahymena toxicity assay could be used as a complementary system to rapidly elucidate the cytotoxic potential of fungicide.

Bacterial Surface Traits Influence Digestion by Tetrahymena pyriformis and Alter Opportunity to Escape from Food Vacuoles.

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide allows any substance carrying amino- or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm.

Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis.

Campylobacter jejuniis the leading cause of bacterial gastroenteritis worldwide. Transmission to humans occurs through consumption of contaminated food or water. The conditions affecting the persistence of C. jejuniin the environment are poorly understood. Some protozoa package and excrete bacteria into multilamellar bodies (MLBs). Packaged bacteria are protected from deleterious conditions, which increases their survival. We hypothesized that C. jejuni could be packaged under aerobic conditions by the amoeba Acanthamoeba castellanii or the ciliate Tetrahymena pyriformis, both of which are able to package other pathogenic bacteria.A. castellanii did not produce MLBs containing C. jejuni In contrast, when incubated with T. pyriformis,C. jejuni was ingested, packaged in MLBs, and then expelled into the milieu. The viability of the bacteria inside MLBs was confirmed by microscopic analyses. The kinetics of C. jejuni culturability showed that packaging increased the survival of C. jejuniup to 60 h, in contrast to the strong survival defect seen in ciliate-free culture. This study suggests that T. pyriformis may increase the risk of persistence of C. jejuniin the environment and its possible transmission between different reservoirs in food and potable water through packaging.

Effect of hexamethylene bisacetamide (HMBA) on the microtubular system of Tetrahymena pyriformis.

HMBA (10-[3-hydroxy-4-methoxy benzylidene)]-9(10H)-anthracenone) is an inhibitor of tubulin polymerization and a developmental inducer in mammalian cells. The effect of HMBA on the microtubular system of Tetrahymena was investigated. This is the first case when its effect was studied in an unicellular animal, by using immunocytochemistry, confocal microscopy, Flutax-1 staining and flow cytometry. In Tetrahymena, HMBA (20 nM.; 10 and 45 min) significantly decreased the label of transversal microtubules (without affecting longitudinal ones) and also decreased the diffuse cytoplasmic fluorescence (tubulin-dimer pool). However, it increased the gross amount of alpha-tubulin and acetylated tubulin. Cilia showed an extraordinary strong labeling. Longer treatments (45 min) were toxic. There is a possibility, that the extremely rich tubulin content of cilia was due to the inducer effect of HMBA or to the self-defense of the cell.

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.

Rapid hydrogen sulfide consumption by Tetrahymena pyriformis and its implications for the origin of mitochondria.

Although sulfide is typically regarded as toxic to eukaryotic cells, it is avidly consumed by Tetrahymena pyriformis. That was observed only when the sulfide concentration was kept below 1 microM. Previously concentrations that were too high had been tested. A new device (Sulfidostat) was used to measure sulfide consumption in steady-state concentrations as low as 10(-12)M. The technique was validated non-biologically by slowly injecting AgNO(3) into buffer and using Ag(2)S precipitation to mimic sulfide consumption, confirming that rates of sulfide consumption could be measured independently of sulfide concentrations. With T. pyriformis, sulfide consumption was 0.25 micromol (gprotein)(-1)s(-1) in 0.5 microM sulfide. Sulfide consumption required O(2) and was inhibited by HCN or by too much sulfide. When cells were separated into fractions, sulfide consumption occurred in the particulate (mitochondrial) fraction. Unexpectedly, the soluble cytosolic fraction slowly produced sulfide even when aerated. The observations are consistent with the conjecture that mitochondria evolved from sulfidotrophic symbionts in a sulfidogenic host cell.

[A cytophotometric study of the influence exerted by epidermal growth factor on RNA and protein synthesis in the ciliate Tetrahymena pyriformis]. / Tsitofotometricheskoe issledovanie vliianiia épidermal'nogo faktora rosta na sintez RNA i belka v infuzoriiakh Tetrahymana pyriformis.

The influence of EGF (10(-8) M) on RNA and protein synthesis in the ciliate Tetrahymena pyriformis was studied. It was found that EGF stimulated RNA and proteins synthesis in T. pyriformis within 3 h after addition of EGF.

Cytotoxicity assessment of three therapeutic agents, cyclosporin-A, cisplatin and doxorubicin, with the ciliated protozoan Tetrahymena pyriformis.

Cyclosporin-A, a drug possessing potent immunosuppressive properties, is used to prevent allograft rejection. Cisplatin and doxorubicin are two of the pharmaceutical drugs most widely used in cancer chemotherapy. In this study, the cytotoxicological impact of these three therapeutic agents was determined using bioassays performed with a unicellular eukaryote, the ciliated protozoan Tetrahymena pyriformis. For this purpose we used the population growth impairment test and the non-specific esterase activities test. We also examined some morphological effects. The results show that these three agents are toxic towards T. pyriformis. A concentration-dependent inhibitory effect on the cell proliferation rate of T. pyriformis populations was found for the three drugs. The IC(50) values were, respectively, 42.03+/-4.64, 124.37+/-7.47 and 74.62+/-6.12 microM for cyclosporin-A, cisplatin and doxorubicin. Non-specific esterase activities were also modified compared with untreated cells. The IC(50) values were, respectively, 88.32+/-8.35 and 44.61+/-3.33 microM for cisplatin and doxorubicin. Exposure of T. pyriformis to these drugs caused the prompt appearance of digestive vacuoles concentrating particulate elements. This phenomenon was more pronounced at higher concentrations. We also observed deformed cells with cisplatin. T. pyriformis bioassays can offer an alternative in vitro method to cell cultures for the risk assessment of potentially toxic drugs.

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.

[Lysozyme-antilysozyme interactions in protozoa-bacteria communities (a model Tetrahymena-Escherichia community)]. / Lizotsim-antilizotsimnye vzaimodeistviia v protozoino-bakterial'nykh soobshchestvakh (na modeli tetrakhimeny-ésherikhii).

Lysozyme and antilysozyme activities present in a wide range of microorganisms determine the so-called lysozyme-antilysozyme system of hydrobionts, which greatly contribute to the formation of aquatic biocenoses. However, the mechanism of the functioning of this system in natural freshwater communities remains obscure. The experimental investigation of lysozyme-antilysozyme interactions in a model Tetrahymena--Escherichia community showed that the antilysozyme activity of Escherichia coli leads to incomplete phagocytosis, thus enhancing bacterial survival in a mixed culture with infusoria. The selection and reproduction of bacterial cells resistant to grazing by infusoria determine the character of host-parasite interactions and allow bacteria to survive. It was demonstrated that the antilysozyme activity of microorganisms, which is responsible for bacterial persistency in natural biocenoses, is involved in the maintenance of protozoa-bacteria communities in bodies of water.

[The ultrastructural characteristics of Francisella tularensis interaction with Tetrahymena pyriformis]. / Ul'trastrukturnye osobennosti vzaimodeistviia Francisella tularensis s Tetrahymena pyriformis.

The electron-microscopic study of the interaction of F. tularensis virulent and attenuated strains with infusoria of the species T. pyriformis was dynamically studied. In this study the structural changes of F. tularensis and T. pyriformis cells, as well as their capacity for survival, were revealed. The data on their ultrastructure correlated with the dynamics of the number of both F. tularensis and T. pyriformis: during the whole term of observation the tendency to a slow decrease in the number of F. tularensis was registered with the concentration of T. pyriformis remaining stable. The interaction of F. tularensis with T. pyriformis may be regarded as a variant of commensal, but not antagonistic interactions.

Effect of glucosphingolipid synthesis inhibitor (PPMP and PDMP) treatment on Tetrahymena pyriformis: data on the evolution of the signaling system.

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) are structural analogues of ceramide; inhibiting UDP-glucose : ceramide glucosyltransferase. After treatment with these synthetic ceramide analogues the expression of glucosphingolipid decreases, while ceramide and sphingomyelin levels increase in the cells of higher eukaryotes. In the unicellular Tetrahymena pyriformis, treatment with PDMP (10-20 microM) and PPMP (40-80 microM) influenced the synthesis of galactose, glucosamine and mannose-containing lipids. On the whole the amount of these lipids was reduced, but new galactose and glucosamine-containing lipids appeared (the exact structures of these lipids were not characterized). Incorporation of (32)P into phosphatidylethanolamine (PE) and phosphatidic acid (PA) was decreased significantly; however the amount of inositol phospholipids were increased. The incorporation of 3H-serine into phosphatidylserine was abolished, but incorporation into sphingomyelin and ceramide was increased. The cytoskeletal elements (silver line system) were disturbed on the basis of scanning electron microscopic pictures. The TRITC-Con A binding and the morphology of the cells were influenced as revealed by confocal laser scanning microscopic analyses. In contrast to higher eukaryotes, in Tetrahymena the shorter fatty acyl chain variant (PDMP) proved to be more effective in each of the examined parameters, while the longer chain variants (PPMP) had milder activity.

Vanadate affects nuclear division and induces aberrantly-shaped cells during subsequent cytokinesis in Tetrahymena.

Sodium orthovanadate at 0.1-5.0 mM affected cell proliferation of Tetrahymena in a dose-dependent manner. At 1 h the cell increment was 76-12% of the control (100%), but after lag periods in 1-5 mM the growth rate remained at 76% of control in 0.1 mM vanadate and at 64-61% of control in 0.2-5.0 mM vanadate. Endocytosis was affected in both a time- and dose-dependent manner; an increasing number of cells did not form vacuoles. Cell motility increased initially in 0.1 mM vanadate but decreased later as it did in 0.5-2.0 mM vanadate where the proportion of immobile cells increased with time. Cell divisions occurred at all concentrations but macronuclear elongation was disturbed and subsequent cytokinesis resulted in daughter cells containing the entire G2 macronucleus, a large or small portion of it, or no nucleus at all. Moreover, odd cell shapes appeared with time. The size of the cell and nucleus increased but there was great variation with disturbed cytoplasm/nucleus ratios. Treated cells had dilated rough endoplasmic reticulum that included dense material, presumed to be vanadate, which was not seen in control cells. Scant amounts of dense material were found in dense granules, small vacuoles, and abundantly in contractile vacuoles. It is argued that interference with proper microtubular function is the main effect of vanadate.

[The interaction of Escherichia coli, possessing the antilysozyme trait, with infusoria]. / Vzaimodeistvie Escherichia coli, obladaiushchikh antilizotsimnym priznakom, s infuzoriiami.

In this work isogenic E.coli strains K-12J53 and J53pAlz60, differing in the presence of the antilysozyme characteristic, were used. In the process of joint cultivation the degradation of lysozyme in protozoa by antilysozyme-active E.coli was established. The initial culture of antilysozyme-active E.coli K-12pAlz60 was found to be slightly heterogeneous with respect to the antilysozyme characteristic; this heterogeneity increased after the interaction of E.coli with protozoa. As the result of their joint cultivation with Tetrahymena, clones with low (2 micrograms/ml) antilysozyme activity (ALA) disappeared from the population and clones with medium and high values of ALA (3, 4, 5, 6 micrograms/ml) accumulated. The dynamics of the interaction of infusoria with Escherichia cells (ALA+) on the ultrastructural level in 1-6 and 24 hours revealed the gradual increase of processes leading to the destruction of most of the bacteria (up to their complete digestion) and, at the same time, the presence, observed also at an early period, of intact bacteria, resistant to lysis, whose survival was ensured by their antilysozyme characteristic.

Transmission electron and atomic force microscopic observation of polysomes on carbon-coated grids prepared by surface spreading.

A simple modification of surface spreading that enables easy examination of subcellular structures by both transmission electron microscopy (TEM) and atomic force microscopy (AFM) was developed in the present study. Specimens were adsorbed onto carbon-coated electron microscopy (EM) grids with parlodion backing for comparison of images obtained using TEM and AFM. Fine structures of ribosomes and polysomes were observed in this study. Washing the mounted specimen with detergent such as 0.05% Photo-Flo 200 before conducting negative staining or metal shadowing brought clear visualization of ribosomes and polysomes. The same preparation could apply to AFM imaging without any additional treatment.

Intracellular insulin binding in Tetrahymena pyriformis.

Insulin, a classic vertebrate hormone, produces alterations in cellular metabolism and growth in the ciliate Tetrahymena pyriformis, as well as an increase in insulin binding upon subsequent exposure, a phenomenon known as hormonal imprinting. An antibody to a peptide corresponding to the alpha-subunit of the human insulin receptor (amino acid residues 657-670) was used to investigate the location and to partially characterize immunoreactive proteins in insulin-exposed and non-insulin-exposed cells (control). Confocal microscopy revealed immunofluorescent labeling of cilia, nuclei, vesicles and an oblong structure of unknown nature. Labeling of nuclei, mitochondria and ciliary microtubules was seen with immunoelectron microscopy. Labeling was absent on the cell and ciliary membranes by immunoelectron microscopy. Polyacrylamide gel electrophoresis revealed several differences in protein composition between control and insulin-exposed ciliary membrane extracts, especially in the 30-50 kDa range. Immunoblotting revealed 2 reactive proteins in whole cell lysates but none were detected in ciliary membrane extracts or wheat germ agglutinin affinity column eluates of T. pyriformis whole cell preparations. Based on these findings it is unlikely that a cell surface structure similar to a mammalian insulin receptor exists in T. pyriformis.

F-actin bundling activity of Tetrahymena elongation factor 1 alpha is regulated by Ca2+/calmodulin.

Translation elongation factor 1 alpha (EF-1 alpha) catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome. Previously, Tetrahymena 14-nm filament-associated protein was identified as EF-1 alpha [Kurasawa et al. (1992) Exp. Cell Res. 203, 251-258]. This and several other studies suggest that EF-1 alpha functions not only in translation but also in regulation of some part of the cytoskeleton. Tetrahymena EF-1 alpha bound to F-actin and induced bundling of F-actin. We investigated the effects of GTP/GDP and Ca2+/calmodulin on F-actin bundling activity of EF-1alpha. The presence of GTP, GDP, or guanylyl-imidodiphosphate (GMP-PNP) slightly decreased the amount of EF-1 alpha which bound to F-actin, but each had virtually no effect on the F-actin bundling activity. The formation of F-actin bundles by EF-1 alpha was Ca(2+)-insensitive. In the absence of Ca2+, calmodulin did not bind to EF-1 alpha and F-actin. On the other hand, in the presence of Ca2+, calmodulin directly bound to EF-1 alpha but did not have any serious influence on EF-1 alpha/F-actin binding. Under the conditions, electron microscopy demonstrated that Ca2+/calmodulin completely inhibited the F-actin bundling by EF-1 alpha. These results indicate that CA2+/calmodulin regulates the F-actin bundling activity of EF-1 alpha without inhibition of the binding between Ef-1 alpha and F-actin.

The relationship of the endoplasmic reticulum to the Golgi complex in Tetrahymena pyriformis.

The endoplasmic reticulum (ER) and the closely connected, single dictyosomal Golgi apparatus of Tetrahymena pyriformis cells showed random distribution in the cytoplasm. Ribosomes were evident, and coated vesicles pinched off from the ER were seen. The membranes of the endoplasmic reticulum generally formed a tube-like structure, although after histamine treatment multiple, folded and circular structures were observed. The number of coated vesicles detaching from the endoplasmic reticulum increased as a result of histamine treatment.

Comparison of axonemal proteins from two kinds of Tetrahymena. I. Different characteristics of dyneins in heat stability.

Tetrahymena thermophila could still swim after incubation of the cell body at 40 degrees C for 30 min, whereas Tetrahymena pyriformis did not show any motility after the treatment. Turbidity measurements revealed that axonemes of T. pyriformis lost ATP-dependent sliding activity by the heat treatment, whereas those of T. thermophilia still had the activity under the same conditions. In connection with this difference in susceptibility to high temperature, the biochemical characteristics of dyneins were compared between the two species of Tetrahymena. Axonemal dyneins from the two species had significant vanadate-sensitive ATPase activity even after the heat treatment. Native gel electrophoresis and the following two-dimensional electrophoresis showed that the outer arm dynein of T. thermophilia is more stable in maintaining native configuration than that of T. pyriformis against the heat treatment, although both treated dyneins keep three (alpha, beta and gamma) subunits. Analysis by peptide mapping demonstrated that beta- and gamma-subunits of the outer arm dynein are considerably different in amino acid sequences between the two species. These results imply that dynein of T. thermophilia changed their amino acid sequences and biochemical characteristics to adapt to high temperature.

Developmental DNA rearrangements and micronucleus-specific sequences in five species within the Tetrahymena pyriformis species complex.

In Tetrahymena thermophila, the development of a transcriptionally active macronucleus from a transcriptionally inert micronucleus includes the elimination of many segments of DNA, the bulk of which belong to repetitive sequence families. Two approaches were used to study the interspecies variations in developmentally eliminated DNA segments. First, the occurrence of restriction fragments crosshybridizing to developmentally eliminated DNA segments isolated from T. thermophila was examined in other species of Tetrahymena. Most micronucleus-specific sequence families examined showed large differences in numbers and intensities of crosshybridizing bands in different species, indicating the possibility of gain or loss of repeats within each of the sequence families. Second, the presence of developmentally excisable DNA segments, i.e., of rearrangement sites, was examined in the same set of species at a number of unique loci. This was carried out by comparing the hybridization patterns of seven unique macronucleus-retained sequences in the micro- and macronuclei of each of the species. Essentially all of the loci displayed variability with respect to the presence of rearrangement sites among the species examined. Results from the two approaches indicate that generation or loss of developmental rearrangements can occur among the species examined here.