Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

The egg-jelly macromolecule,a fucose sulphate glycoconjugate,originates from the accessory cells of the ovary in the sea urchin Hemicentrotus pulcherrimus

Summary The immunocytochemical localization of the egg-jelly macromolecule, a fucose sulphate glycoconjugate (FSG) that induces the acrosome reaction in spermatozoa, was investigated in ovaries of the sea urchin Hemicentrotus pulcherrimus by use of a polyclonal antibody. The polyclonal antibody reacted with the accessory cells and oocytes in the ovarian lumen. In the accessory cells, evidence of an intense immunohistochemical reaction was observed in many globules of variable density. Products of the specific immunohistochemical reaction were frequently observed in the surface region of oocytes, at a distance from the ovarian wall. At the ultrastructural level, the polyclonal antibody was found to react with the material present in the vacuole-like structures of the globules in the accessory cells. Many gold particles, demonstrating specific immunolabelling, were associated with well-developed microvilli on the vitellogenic oocytes. In the mature oocytes, intense labelling was observed in the jelly coat but not in the vitelline coat. By contrast, oogonia and early oocytes were barely labelled. Quantitative data indicated that the extent of immunolabellings in the surface region of oocytes was very high in the vitellogenic and mature oocytes. In all cases, neither the oocyte cytoplasm nor the subcellular organelles were labelled. These results suggest that FSG is produced by the accessory cells and is deposited initially on the surface of vitellogenic oocytes for the formation of jelly. These findings may provide a new insight into the role of the accessory cells in the reproductive process of the sea urchin. Offprint requests to: N. Suzuki     

Uridine uptake in a unicellular eukaryote during the interdivision period and after growth arrest

The rate of uridine uptake was measured in Tetrahymena after shiftdown to non-nutrient physiological salt solution. Uptake follows Michaelis-Menten kinetics and an apparent K_m of transport of 2 × 10⁻⁶ M has been estimated. This value is in good agreement with those reported for tissue-derived cells in culture. Incorporation of uridine into RNA follows similar kinetics suggesting that uptake is rate limiting for incorporation. Within three hours after shiftdown the rate of uptake is decreased by an order of magnitude. Also at three hours after shiftdown pairing occurs between cells of complementary mating types. It seems likely that the change in uptake is a reflection of a surface change associated with differentiation. The rate of uptake was also measured during the interdivision period using cells synchronized by a physical selection procedure. A change in rate occurs at the time the cells begin replication of DNA and is essentially stable thereafter. These results indicate that there exists in Tetrahymena a relationship between surface properties as assayed by uridine uptake and properties of growth and differentiation.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Phosphonoacetic Acid: Effect on and Disposition by Tetrahymena1

The antiviral agent phosphonoacetic acid inhibits growth of Tetrahymena thermophila at concentrations comparable to those inhibiting growth of other eukaryotic cells, with 50% inhibition at 0.5 mM phosphonoacetic acid. The compound is cytotoxk to Tetrahymena at concentrations greater than 2.0 mM. When a culture of Tetrahymena the growth of which was totally inhibited by 2.0 mM phosphonoacetic acid was diluted with fresh medium, growth resumed in an exponential, rather than synchronous, fashion. [2–¹⁴C]phosphonoacetic acid is not metabolized by Tetrahymena.     

Effects of inhibitors of RNA and protein synthesis on activation of glyconeogenesis in Tetrahymena

The effects of actinomycin D, puromycin, and p-fluorophenylalanine on the activation of glyconeogenesis in Tetrahymena were studied. The extent of activation of glyconeogenesis in cultures containing inhibitor was as great as or greater than in the controls, as was the uptake of tracer levels of acetate into glycogen. These increases occurred despite a partial or complete inhibition of synthesis of isocitrate lyase, a glyconeogenic enzyme in Tetrahymena. Washed cells from these cultures could convert tracer or substrate levels of acetate to glycogen at enhanced rates. When glyconeogenesis was activated in starved cells in the presence of inhibitor, there was a negligible increase in the amount of isocitrate lyase, but a significant increase in the rate of glyconeogenesis. The data indicate that glyconeogenesis in Tetrahymena can be activated in the absence of enzyme synthesis.     

Generation and characterization of novel antibodies highly selective for phosphorylated linked histone H1 in Tetrahymena and HeLa cells

Phosphorylated forms of Tetrahymena macronuclear histone H1 were separated from each other and from dephosphorylated H1 by cation-exchange HPLC. A homogeneous fraction of hyperphosphorylated macronuclear H1 was then used to generate novel polyclonal antibodies highly selective for phosphorylated H1 in Tetrahymena and in human cells. These antibodies fail to recognize dephosphorylated forms of H1 in both organisms and are not reactive with most other nuclear or cytoplasmic phosphoproteins including those induced during mitosis. The selectivity of these antibodies for phosphorylated forms of H1 in Tetrahymena and in HeLa argues strongly that these antibodies recognize highly conserved phosphorylated epitopes found in most H1s and from this standpoint Tetrahymena H1 is not atypical. Using these antibodies in indirect immunofluorescence analyses, we find that a significant fraction of interphase mammalian cells display a strikingly punctate pattern of nuclear fluorescence. As cells enter S-phase, nuclear staining becomes more diffuse, increases significantly, and continues to increase as cells enter mitosis. As cells exit from mitosis, staining with the anti-phosphorylated H1 antibodies is rapidly lost presumably owing to the dephosphorylation of H1. These immunofluorescent data document precisely the cell cycle changes in the level of H1 phosphorylation determined by earlier biochemical studies and suggest that these antibodies represent a powerful new tool to probe the function(s) of H1 phosphorylation in a wide variety of eukaryotic systems. Received: 3 August 1993; in revised form: 9 November 1993 / Accepted: 23 November 1993     

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. Copyright © 2009 John Wiley & Sons, Ltd.     

Phospholipase D in Tetrahymena: activity and significance

We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. I. Fine Structure and Cytochemistry*†

SYNOPSIS. An extracellular surface coat was observed at the fine-structural level on the outer lamina of the pellicular and flagellar membranes of intact Trypanosoma musculi bloodstream forms. The surface coat had a mean width of 9.2 nm, and was composed of a somewhat electron dense, uneven, fibrillar-like matrix. Brief trypsin treatment of living blood forms completely removed the cell surface coat. Several cytochemical methods applicable to electron microscopy were used to detect the presence and distribution of carbohydrates in the trypanosome's surface coat and pellicular membrane. The polycationic dye compounds employed were: ruthenium red, ruthenium violet, Alcian blue chloride, and lanthanum nitrate. Electron-dense stain reaction products, indicative of polysaccharides, were evident in the surface coat of cells treated with these dyes, which also agglutinated both living and glutaraldehyde fixed cells. Like the surface coat, the pellicular membrane of trypsinized cells gave strong positive staining reactions with the several dyes, indicating the presence of membrane bounded carbohydrates, and living and glutaraldehyde-fixed trypsinized cells were agglutinated with the polycationic stains. Bloodstream forms were treated with the enzymes, α-amylase, dextranase, and neuraminidase. No obvious morphologic difference, however, was apparent between the surface coat of untreated cells and those subjected to treatment with any of the various glycoside hydrolase enzymes. Further, these enzymes had no apparent gross effect on the staining affinity of the surface coat for the several polycationic dyes. Cationized ferritin was used to visualize the negative cell surface charge of T. musculi bloodstream forms. Large quantities of cationized ferritin were bound in the surface coat matrix. Glycoside hydrolase enzyme treatments had no apparent effect on the amount of ferritin bound in the surface coat. Cationized ferritin was bound also to the outer lamina of the pellicular membrane in trypsinized cells, which had quantitatively less ferritin bound per surface unit area than bloodstream forms untreated by the enzyme. Living and glutaraldehyde-fixed cells were agglutinated with cationized ferritin. The results obtained in the various experiments indicated that polyanionic polysaccharides were constituent terminal ligands of the surface coat matrix and pellicular membrane in T. musculi bloodstream forms.     

Cytolytic Activity Released from Tetrahymena1

Using a hemolytic assay system, we have detected cytolytic activities in extracellular medium from Tetrahymena thermophila and T. pyriformis. In addition, we have identified two phospholipase activities (types A and C) from the same source by thin layer chromatography analysis of the breakdown products of a phospholipid preparation. The hemolytic activity peaks at low pH values. It is inhibited by egg lecithin, supporting the view that a phospholipase activity is involved in the cytolytic processes. Cytolytic activities may play important roles in Tetrahymena, both in nutrition, especially in parasitic and scavenger forms, and in defense against predators. Tetrahymena is probably partly protected from its own released cytolytic phospholipase by having a high proportion of phosphonolipids on its surface membrane.     

Cilia regeneration in Tetrahymena : A simple reproducible method for producing large numbers of regenerating cells

We have developed an improved medium in which Tetrahymena can be deciliated by gentle shearing. The cells remain viable and regenerate a new complement of cilia. Unlike previous methods for viable deciliation of Tetrahymena, this method is easily adaptable to large numbers of cells, to cells in different stages of the life cycle (growing, starved, conjugating), and to both commonly studied species, T. thermophila and T. pyriformis. Starved T. thermophila deciliated by this method regained motility by 1 h, regenerated oral apparatus by 4.0 h and restored tubulin in cilia at a linear rate of about 3 pg h⁻¹ cell⁻¹.     

Ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans (Müller) (mollusca,prosobranchia) and the ommatophore of Helix pomatia L. (Mollusca,Pulmonata)

The ultrastructure of the supporting cells in the chemoreceptor areas of the tentacles of Pomatias elegans and Helix pomatia is very similar. Complex apical structures are present, and the lateral plasma membrane exhibits three zones: (1) a zone of slight interdigitations; (2) a zone characterized by longitudinal plicae; (3) a zone of basal radiculae. The portions of the sensory cells located within the epithelial layer are accommodated in longitudinal grooves in the supporting cells. However, there are also differences. In Pomatias elegans the apical surface is differentiated into long microvilli that are sometimes dichotomously branched and invested by a surface coat along their entire length. Cytofilia and cilia of the sensory cells pass through this layer of microvilli and surface coat throughout its entire width. In Helix pomatia the supporting cells are somewhat smaller and the apical differentiation consists of candelabra-like protrusions, which are usually three times dichotomously branched. The final branchings, corresponding to microvilli, are called terminal twigs. They are covered by a surface coat, which forms a feltwork. The cytofilia and cilia of the sensory cells that intertwine among the protrusions are confined to the space below the terminal twigs, where they compose the spongy layer.     

Barcodes Reveal 48 New Species of Tetrahymena,Dexiostoma, and Glaucoma: Phylogeny,Ecology, and Biogeography of New and Established Species

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena‐like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well‐established “australis” or “borealis” clades; the minority forms a divergent “paravorax” clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self‐splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the “australis/borealis” split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms.     

Is cyclin Zeuthen's “division protein”?

Biochemical evidence is presented for the presence of cyclin in Tetrahymena. Zeuthen previously postulated the existence of a heat-labile “division protein” to explain heat-shock-induced division synchrony in Tetrahymena [(1964) Synchrony in Cell Division and Growth (Zeuthen, E., Ed.), pp. 99–158, Interscience, New York]. We show that cyclin is heat-labile in Tetrahymena and suggest that cyclin may be Zeuthen's division protein. Cyclin and cell cycle control is of interest in Tetrahymena because the division mechanism drives macronuclear amitosis, closed and acentric micronuclear mitosis, and cortical differentiation in this cell type.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Further Studies of Dopamine Metabolism and Function in Tetrahymena1

The large amounts of dopamine accumulated by cells of Tetrahymena pyriformis strain NT-1 and secreted into their growth medium were found to depend primarily upon an extracellular, non-enzymatic conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA); L-DOPA was then rapidly taken into the ceils and transformed into dopamine enzymatically. Efforts to find physiologically significant dopamine binding sites on the cell surface or dopamine-sensitive adenylate cyclase activity were unsuccessful, suggesting that the catecholamine does not function in Tetrahymena as it does in higher animals.     

Use of Sequence-Specific Antisense Oligodeoxynucleotides to Determine the Protozoan Parasite Antigen Recognized by Nonspecific Cytotoxic Cells

The antigen on the protozoan parasite Tetrahymena pyriformis recognized by catfish nonspecific cytotoxic cells (NCC) is a 46- to 48-kDa protein referred to as NKTag. The complete cDNA-derived amino acid sequence of NKTag has been obtained. The antigenic determinant of NKTag corresponding to the NCC binding site has been determined with synthetic peptides in target cell competition experiments. To more directly characterize the mechanism of parasite:effector cell interaction, we applied NKTag sequence-specific antisense oligodeoxynucleotides to Tetrahymena in vitro. NKTag mRNA translation by Tetrahymena was blocked by specific antisense (AS) oligodeoxynucleotides. 5′-3′ sense (S) oligodeoxynucleotide sequences were synthesized corresponding to the first 17 N-terminal amino acids of NKTag (in addition to −2 untranslated codons plus the start codon). Complimentary AS oligodeoxynucleotides were likewise synthesized. To determine the optimum in vitro conditions for AS treatment, we tested parasites at various phases of their growth cycle for the effects of a single AS treatment. At 9 h post-AS treatment (during the linear phase of the growth curve), maximum reduction in membrane expression of NKTag was observed. Eighty-five percent of Tetrahymena were positive for expression of NKTag at 0 time post-AS treatment versus 13% positive at 9 h. Membrane expression of AS-treated parasites returned to normal levels by 24 h post-treatment. In cold target inhibition experiments, the reduced NKTag expression by Tetrahymena at 9 h AS treatment was confirmed by observing a complete inability (compared with S-treated parasites) to compete with IM-9 cells for binding with NCC. These data demonstrated a unique experimental in vitro system to define the antigen determinant on target cells responsible for recognition by cytotoxic effector cells that participate in innate immune responses. Received: 14 June 1999 / Accepted: 4 September 1999     

Hereditary modification of Tetrahymena resulting in polymorphic cells : Effects of heat shocks and cellular volume

Exposure of a Tetrahymena culture to numerous heat shocks of the type used to synchronize cell divisions results in cells having a volume up to 10 times that of untreated cells. Besides, a few percent of the cells are hereditarily modified and give rise to lines of monstrous polymorphic cells. To facilitate production and isolation of monstrous cells a heat shock program combined with a selection procedure and a shortened genetic dilution test has been tested. All treatments known to produce the modified cells initially result in cellular growth without accompanying divisions. It is shown that increase in cellular volume as produced by the heat shocks is not sufficient to induce the modifications. It is suggested that multiplication of cortical cytoplasmic structures or morphogenetic reference points may be essential. Some similarities between the hereditarily modified Tetrahymena and cancer cells are mentioned together with the possibility that hereditary changes may sometimes be induced by conditions generally considered unable to do so.