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°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 (α, β and γ) subunits. Analysis by peptide mapping demonstrated that β- and γ-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.     

Three New “Biological” Species of Tetrahymena (T. hegewischi n. sp., T. sonneborni n. sp., T. nipissingi n. sp.) and Temperature Tolerance of Members of the “pyriformis” Complex1

Stocks of the Tetrahymena pyriformis complex have been collected in North America and their mating reactivity has been studied. In addition to stocks mating with Tetrahymena americanis, T. borealis, T. pigmentosa, T. hyperangularis, and T. australis, stocks belonging to old syngen 5 and three new mating groups, numbers 13, 14, and 15, were discovered. Syngen 5 and groups 13 and 14 are distinct “biological” species, based on their reproductive isolation from other groups and on the ability of withingroup crosses to produce immature progeny. These species have been named T. hegewischi n. sp., T. sonneborni n. sp., and T. nipissingi n. sp., respectively. The cross between the two group 15 stocks did not produce immature progeny, and there is not sufficient evidence to conclude that this pair of stocks represents a separate species. Temperature tolerance measurements have been made on stocks representing all known micronucleate members of “pyriformis” complex. Within each species, the range of temperature tolerances is narrow; the average within-species standard deviation is 0.63°C. The species averages range from 32.7 to 40.7°C. Using syngen numbers, the order from lowest to highest temperature tolerance is 9, 8, 10, 7, 6, 4, 13, 14, 12, 11, 5, 3, 2, 1. The large differences among species make temperature tolerance a useful aid in identification, but the origins of the differences among species are unknown.     

Survival of Tetrahymena pyriformis and Paramecium aurelia Following Freezing*

SYNOPSIS. Tetrahymena pyriformis strains E, A-136 31C and IMT II survived freezing in 10% dimethylsulfoxide when the temperature was lowered to freezing at 4.5 C/min. Survival was then obtained for at least 128 days by lowering the temperature rapidly to 95°C. Of the 3 strains, T. pyriformis IMT II was most resistant to the effects of freezing. Its volume averaged about half that of either of the other strains and may have contributed to the differences in survival. In addition to differences among strains, a medium relatively low in the concentration of nutrients, a culture nearing peak population, and a rate of cooling of 4.5 C/min, all gave best survival. Paramecium aurelia regained motility after being frozen in 6 to 7.5% dimethylsulfoxide for as long as 7 days at either –27 or –196 C, but cultures were obtained only after storage for 20 min at –27 C. A concentration of 6 to 7.5% dimethyl-sulfoxide, cooling at 4.5 C/min, and culture media containing Aerobacter aerogenes or composed of a commercially available composition were all required for survival of P. aurelia.     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

A Multi-Unit Sampling System and Its Use in the Characterization of Ultradian and Infradian Growth in Tetrahymena*

SYNOPSIS. A multi-unit automatic sampling device for investigation of microbial growth under a wide variety of conditions is described. The kinetics of asynchronous population growth for batch cultures of Tetrahymena pyriformis (W) at several temperatures show that there are 2 distinct growth phases: an exponential ultradian growth phase that is strongly temperature dependent and a non-stationary growth phase, the infradian phase, that shows little or no temperature dependence over the range from 15–27 C.     

An Immobilization Antigen in Tetrahymena pyriformis Expressed Under Conditions of High Salt Stress*

SYNOPSIS. Cells of Tetrahymena pyriformis syngen 1 exposed to 200 mM NaC1 without previous exposure to a saline medium die within minutes. Lines that have been adapted to higher salt concentrations by growing in axenic peptone medium with 100 mM NaC1 are able to grow in previously intolerable concentrations. Acclimation to higher salt concentrations is accompanied by a transformation from the H serotype, expressed at room temperature in the standard peptone medium, to a new serotype, St. Complete transformation occurs at concentrations approaching the LD₅₀ for nonadapted cells, and the time required for the transformation is 3–4 days. The reverse transformation, St to H, involves a time interval during which the cells are refractory to both anti-St and anti-H. Among 12 of the 13 inbred families of T. pyriformis syngen 1, 2 different high salt serotypes are found. One of these, Sta, is found among families A, Al, A3, B, D, D1, E, and F. The other, Stc, occurs in families C, C1, C2, C3, and possibly B2.     

The effect of temperature on nucleotide pool formation in Tetrahymena pyriformis

Warming of exponentially growing T. pyriformis to 34°C results in severe inhibition of nucleotide pool formation. The utilization of the pool for stable RNA synthesis is poorly affected at the high temperature. It thus appears that the synthesis and processing of ribosomal RNA precursors are not primarily impaired at 34°C.     

Growth Kinetics of Three Species of Tetrahymena On Solid Agar*

A nutrient-agar method without liquid overlay has been developed for cultivation of ciliates. Three species of Tetrahymena-T. pyriformis strain W, T. rostrata strain UNI, and T. vorax strain V₂S, representing the 3 main groups of Tetrahymena species, were used; however the method should apply to other ciliates. Growth on the surface of the agar was facilitated by an optimal surface-to-volume ratio yielding a high density of ciliates (5.8 × 10₅ cells/cm² for T. pyriformis at 25 C) and short generation times (3 h for T. pyriformis at 30 C). At the highest density achieved, the cells became irregularly hexagonal and formed a monolayer “tissue” on the agar. Ciliates grown on agar were like those in liquid culture, typical oral ciliature, food-vacuole formation, and typical cortical patterns being retained. Advantages of this method include high cell density, easy recovery, and optimal O₂ supply. the organisms can also be cultivated on the surface of sterile cellulose-nitrate filters, facilitating in situ fixation and staining as well as transfer into different media by transfer of filters with cells, without prior centrifugation and resuspension.     

The 5S and 5.8S Ribosomal RNA Sequences of Tetrahymena thermophila and T. pyriformis1

The nucleotide sequences of the 5S rRNAs of Tetrahymena thermophila and two strains of T. pyriformis have been determined to be identical. The 5.8S rRNA sequences have also been determined; these sequences correct several errors in an earlier report. The 5.8S rRNAs of the two species differ at a single position. The sequencing results indicate that the species are of recent common ancestry. Molecular evidence that has been interpreted in the past as suggestive of an ancient divergence has been reviewed and found to be consistent with a T. pyriformis complex radiation beginning approximately 30–40 million years ago.     

Effects of natural amino acids and sugars on activity of infusiorian cyclases

Natural amino acids and sugars in unicellular 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, tyramine, 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 rose 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 the 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 specter of comparatively simple natural molecules has a hormone-like action.     

Effect of cocaine and crack on the ploidy status of <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>: a study using DNA image analysis

The effect of cocaine and crack on the ploidy status of Feulgen-stained Tetrahymena pyriformis macronuclei using computerized DNA image analysis system was tested. For this purpose, selected doses of 5, 10 and 20 μg (per mL culture) of both drugs were administered for 2, 5 and 20 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the following parameters: Ploidy Balance (PB), Degree of Aneuploidy (DA), skewness and kurtosis. The results have shown a positive effect of both cocaine and crack on PB and on DA of T. pyriformis macronuclei. In particular, our results reveal that the aneugenic effect (which is expressed as a decrease in PB and an increase in DA) of cocaine on T. pyriformis macronuclei follows a dose-dependent manner, while crack induces aneuploidy in a dose-independent manner. Changes in the PB and DA values would induce a disturbance in the cellular density and heterogeneity of chromatin and the increase in skewness and kurtosis values after exposure of T. pyriformis to both drugs, did confirm this hypothesis. These observations were further correlated with alterations in the chromosomal segregation and with damage in mitotic spindle microtubules observed previously. In this study the impact of cocaine and crack on genomic instability and carcinogenesis was further supported and T. pyriformis can be proposed as a model organism to test the nuclear ploidy status after exposure to harmful chemicals and drugs.     

IDENTIFICATION OF LECTINS IN THE KINETIDS OF TETRAHYMENA PYRIFORMIS

Previously we described lectin-like molecules in the ciliate Tetrahymena pyriformis; by application of synthetic neoglycoconjugates it is now shown that T. pyriformis contains considerable amounts of both a β-d-glucose- and a lactose-specific lectin. No evidence for the presence of α-d-mannose-, α-d-galactose- or of α-l-fucose-specific lectins could be obtained. The two lectins, identified in T. pyriformis, are associated with the kinetids. During cell division the lectins disappear or become masked in the fission furrow. Therefore, we assume that these lectins are involved in the organization of the distribution pattern of the kinetids during cell division perhaps due to lectin—glycoprotein interactions.     

Particle-Based Axenic Media for Tetrahymenids*

SYNOPSIS. Autoclavable, natural particulate media simplify axenic cultivation of tetrahymenid ciliates and presumably favor selection for phagotrophy. Viability is at least 2 months at room temperature (24–26 C) for the lipid-sensitive tetrahymenids Tetrahymena setosa, T. corlissi, T. paravorax, T. limacis, and T. patula, also for T. rostrata and (at 12 C), for strains of the T. pyriformis complex and Glaucoma chattoni. A typical medium consists of crude soy “lecithin”+ skim milk powder +Saccharomyces cerevisiae cells. Other useful particules readily available commercially are: whole liver powder, cells of Micrococcus lysodeikticus and Escherichia coli, and powdered residue of liver which had been extracted with 70% ethanol (liver #2). Preliminary experiments indicate that some of these media are suitable for the maintenance of Paramecium octaurelia stock 299S and Colpidium campylum. Such mixtures may serve as points of departure for devising media for more fastidious phagotrophs.     

Thymidylate Synthetase as a Chemotherapeutic Target in the Treatment of Avian Coccidiosis*

SYNOPSIS. Thymidylate synthetase (E.C.2.1.1.45) has been demonstrated in unsporulated oocysts of Eimeria tenella. The properties of this enzyme have also been investigated in Tetrahymena pyriformis, as a protozoan model, and 7-day-old chick embryo, as a host model. The enzymes from E. tenella and chick embryo were inhibited by all concentrations of MnCl₂ and MgCl₂ tested. Tetrahymena pyriformis thymidylate synthetase was stimulated by low concentrations of both these cations but was inhibited by high concentrations. Subsequent data refer to chick embryo, E. tenella and T. pyriformis respectively: the apparent K_m was 5.89 μM, 5.94 μM, and 0.53 M for the substrate dUMP: and 5.13 μM, 1.10 μM and 4.65 μM, respectively for the cofactor N⁵N¹⁰-methylenetetrahydrofolate. The pH optimum for the enzyme from both chick embryo and T. pyriformis was 8.0, with Tris-HCl buffer; activity of E. tenella thymidylate synthetase was still increasing at pH 8.2. The E. tenella enzyme was found to have a molecular weight of 4.6–4.9 × 10⁵ daltons. The effects of nucleotides, inhibitors, and the omission of assay components on each enzyme are presented. Thymidylate synthetase from E. tenella is not greatly different from that of chick embryo, but does not resemble the enzyme from T. pyriformis. A case for using thymidylate synthetase as a chemotherapeutic target in the treatment of Eimeria infections remains. Indeed Eimeria may be considered as a model for infections caused by other protozoan parasites, such as Toxoplasma and Plasmodium, provided that suitable inhibitors can be found that are not toxic to the host.     

A hydrogen-bonding network formed by the B10–E7–E11 residues of a truncated hemoglobin from <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> is critical for stability of bound oxygen and nitric oxide detoxification

Abstract  

Truncated hemoglobins (trHbs) are distributed from bacteria to unicellular eukaryotes and have roles in oxygen transport and nitric oxide detoxification. It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein. The O₂ association and dissociation rate constants of T. pyriformis trHb were 5.5 μM⁻¹ s⁻¹ and 0.18 s⁻¹, respectively. The autooxidation rate constant was 3.8 × 10⁻³ h⁻¹. These values are similar to those of HbN from Mycobacterium tuberculosis. The three-dimensional structure of an Fe(II)–O₂ complex of T. pyriformis trHb was determined at 1.73-? resolution. Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound O₂ molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues. Mutations at Tyr25, Gln46, and Gln50 increased the O₂ dissociation and autooxidation rate constants. An Fe(III)–H₂O complex of T. pyriformis trHb was formed following reaction of the Fe(II)–O₂ complex of T. pyriformis trHb, in a crystal state, with nitric oxide. This suggests that T. pyriformis trHb functions in nitric oxide detoxification.     

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.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

Respiratory energy losses related to cell weight and temperature in ciliated protozoa

Summary Respiratory energy losses in five species of ciliated protozoa, Tetrahymena pyriformis Ehrenberg, Vorticella microstoma Ehrenberg, Paramecium aurelia Ehrenberg, Spirostomum teres Claparède and Lachmann and Frontonia leucas Ehrenberg, were investigated at 8.5° C, 15° C and 20° C using Cartesian diver microrespirometry. Q ₁₀ values of 1.15–2.24 were found for four of the species between 8.5–15° C, while in S. teres a Q ₁₀ of 12.98 occurred between these temperatures. Between 15–20° C T. pyriformis and P. aurelia had Q ₁₀ values of 3.73 and 1.56, respectively. Linear double log regressions of oxygen consumption vs. dry weight were derived at each temperature and regression coefficients (b) of 0.2723 (8.5° C), 0.4364 (15° C) and 0.4171 (20° C) were obtained. The results are explained and discussed in relation to previous work on the energetics of ciliated protozoa.     

Mirex and Aroclor® 1254: Effect on and Accumulation by Tetrahymena pyriformis Strain W*

Effects of 2 toxicants, Mirex and Aroclor 1254, on Tetrahymena pyriformis strain W in axenic cultures were investigated. Mirex is a chlorinated hydrocarbon effective against the fire ant, and Aroclor 1254 is a compound structurally related to DDT and used extensively in various industrial processes. Both toxicants reduced growth rates and population densities of T. pyriformis grown at 26 C generally in proportion to concentrations of the chemicals, their effects becoming statistically significant (P < 0.05) at 0.9 μg/liter for Mirex and 1.0 and 10.0 μg/liter for Aroclor 1254. Ciliates exposed to the toxicants for 7 days concentrated Mirex 193 × and Aroclor 60 × as compared to the initial concentrations of these compounds. It is suggested that the chief effect of the 2 toxicants on populations of T. pyriformis and of similarly responding ciliates in nature would be to reduce the availability of these protozoa as food organisms and nutrient regenerators. The ability of the ciliates to concentrate the tested compounds would permit the toxicants to enter into and to be translocated through aquatic food chains. In this manner the compounds could exert toxic effects at higher trophic levels.