The Effects of Purine and Pyrimidines Upon Transformation in Tetrahymena vorax,Strain V2S1

Column chromatography with Biogel P2 (molecular exclusion of 1800 daltons) indicates that the transforming principle causing microstomes to become macrostomes is a small molecule. Absorbance tests show that only those fractions with high absorbance at 260 nm have biological activity, indicating that the active principle is a component of nucleic acids. Tests of purines and pyrimidines show that purines are active, with hypoxanthine having the highest activity. The combination of hypoxanthine with uridine shows a synergistic reaction. As these two compounds are the natural catabolic excretory products from nucleic acids in Tetrahymena, the fact that they induce transformation in concentrated, starving cells may be a survival mechanism allowing cannibalism to be induced when nutrients are depleted, thereby allowing the survival of the transformed cells until such time as adequate nutritional conditions are restored.     

An Analysis of Macrostome Production in Tetrahymena vorax Strain V2S-Type*

SYNOPSIS. Tetrahymena vorax is a small polymorphic holotrichous ciliate capable of transforming to macrostomes when microstomes are washed and suspended with prey in distilled water. Extrinsic factors having an effect on this transformation were examined; maximum yields of macrostomes (in excess of 90%) were obtained under the following conditions: populations of both prey, T. pyriformis, and potential predator, T. vorax microstomes, were grown on Loefer's medium for 48 hours prior to washing in distilled water. The density of the prey was adjusted to 300,000 cells/ml and the predator density to 2,000–3,000 cells/ml. Five ml of prey suspension and 5 ml of T. vorax microstome suspension were mixed together in a large petri dish because a high surface-volume ratio is important for high yields of macrostomes. The pH was adjusted to 6.0 and the petri dish was placed at 20 C for 12 hr. Macrostomes then appeared about 6 hr after addition of the prey. A dialyzable, heat stable substance released by the prey which can induce the microstome-macrostome transformation was isolated. This material was effective after being stored for weeks in the cold; its activity was not affected by the protein digesting enzymes pepsin or trypsin. This factor was called stomatin because its first visible effect in producing microstome-macrostome transformation appeared to be to incite reorganization of the oral structures.     

Evidence for early signaling events in stomatin-induced differentiation of Tetrahymena vorax

The mechanism of stomatin-induced differentiation of Tetrahymena vorax was investigated by in vivo protease degradation of cell surface proteins, the direct measurement of products formed from the activation of phospholipase C, and the use of an array of signal transduction inhibitors/activators. The data indicate that a surface-exposed protein is required for stomatin to signal the cells to differentiate and that the cells are committed to the differentiation pathway within two hours after exposure to stomatin. Analysis of radiolabeled polyphosphoinositols and inositol lipids from control and stomatin-treated populations in the presence of 10 mM LiCl were consistent with a rapid activation of phospholipase C. Within five min following addition of stomatin, this resulted in an increase in polyphosphoinositols and a concomitant decrease in the relative amounts of phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate.     

Lipid modification during cytodifferentiation of Tetrahymena vorax. Whole cell phospholipids and triacylglycerols of microstomal and macrostomal phenotypes

Microstomal cells of the ciliate Tetrahymena vorax V2S can be induced to undergo cytodifferentiation to form an alternate phenotype known as the macrostomal cell; however, sublines of T. vorax exist that respond differently to methods that induce macrostomal cell formation. The phospholipid- and triacylglycerol-bound fatty acid compositions of microstomal and macrostomal cells of a high-transforming subline (designated 3-C) were determined and compared to similar data from cells of a low-transforming subline (designated Ala). Differences in fatty acid composition were found between the two phenotypes as well as between the different sublines. Some change in the distribution of radioactive acetate and lauric acid into phospholipid classes of the different subline was observed, and evidence was also obtained that indicated changes in the relative amounts of the sterol-like pentacyclic triterpenoid tetrahymanol. A limited analysis of the lipid composition of stomatin revealed the presence of small amounts of tetrahymanol, phospholipid and free fatty acid. Stomatin is the naturally produced material obtained from T. pyriformis that triggers differentiation in T. vorax. The existence of a low-transforming subline provides a powerful experimental tool for elucidating the underlying biochemical and molecular mechanisms that control cytodifferentiation in T. vorax and possibly in other eukaryotic cells.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Oral Apparatus Structure in the Carnivorous Macrostomal Form of Tetrahymena vorax

The structure of the oral apparatus in the carnivorous macrostomal form of Tetrahymena vorax has been investigated using serial thin sections and preparations of isolated oral apparatuses. The cilia of the oral apparatus are organized into an undulating membrane that borders the right and part of the posterior margin of the buccal cavity and three membranelles that project from plateaus on the anterior surface. Each membranelle consists of one short row and two longer rows of hexagonally packed kinetosomes. The organization of the microtubules of the oral ribs is identical to that in the T. vorax microstomal cell type. However, the first oral rib originates near the first kinetosome at the anterior end of the undulating membrane. The fine filamentous reticulum that underlies part of the oral ribs in the macrostomal cell type is not striated, unlike the reticulum in the microstomal form. A band of filaments similar to the fine filamentous reticulum extends around the anterior margin of the large cytostomal opening that occupies most of the posterior part of the oral cavity. The single row of microtubules along the left side of the oral cavity and cytostome also has filaments associated with it. A major difference between the microstomal and macrostomal forms in the structure of the oral apparatus is in the oral connectives. The macrostomal cell type contains only a single cross-connective that joins the three membranelles and the anterior portion of the undulating membrane. The posterior or peripheral connective between the posterior ends of membranelles one and two and the posterior end of the undulating membrane is absent.     

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately ?30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca²⁺ spikes with a maximum rate of rise of 7.5 Vs^?1 in microstome and 4.7 Vs^?1 in macrostome cells. Depolarizing voltage steps from a holding potential of ?40 mV induced an inward Ca²⁺ ‐current (I_ca) peaking at ?10 mV, reaching approximately the same value in microstome (?1.4 nA) and macrostome cells (?1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I_Ca in these morphs supports the notion that the voltage‐gated Ca²⁺ channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na⁺‐free solution and was only partially inhibited by extracellular Cs⁺. The inward rectification was completely blocked by replacing Ca²⁺ with Co²⁺ or Ba²⁺ in the recording solution, and is probably due to Ca²⁺ ‐activated inward K⁺ current secondary to Ca²⁺ influx through channels activated by hyperpolarization.     

Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila.

A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila. This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B. The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits. Electrophoresis, immunoblot, and [35S]methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons. An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C. This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions.     

Microstome-Macrostome Transformation in Tetrahymena vorax Strain V2 Type S Induced by a Transforming Principle, Stomatin

SYNOPSIS. Microstome →macrostome transformation in Tetrahymena vorax was induced by suspending microstomes in a transforming principle, stomatin, released by a potential prey, T. pyriformis. It was found that 70–90% of the microstomes formed macrostomes within 7 hours following suspension in this transforming principle. Macrostome formation occurred by the process of oral replacement. This process involved resorption of the microstome oral apparatus and its replacement with a larger (macrostome) one, which arose from an anarchic field that formed behind the resorbing oral area. Ninety-five percent of those microstomes which were destined to form macrostomes were in some stage of oral replacement 195 minutes after their suspension in stomatin. Several commercially produced products were tested over a wide range of concentrations to determine their ability to act as an inducer of macrostomes. Only 2, Trypticase and Bactocasitone, had any activity, and it was too small to be considered really effective. An attempt was also made to destroy the activity of stomatin by using enzymes. RNAse was effective but only in very high concentrations, so it was suggested that this activity might be related to the destruction of RNA within the transforming cell and not related to hydrolysis of stomatin. None of the other enzymes tested had any effect in reducing the activity of stomatin.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange.

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately -55 mV and was blocked by 1 mmol l(-1) TEA. The rate of rise of electrically evoked Ca(2+)-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca(2+) concentration. This suggests that the hyperpolarisation may be caused by activation of Ca(2+)-sensitive K(+) channels. The depolarisation was abolished in Ca(2+)-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca(2+) influx through anterior channels, while Ca(2+) released from intracellular stores activates K(+) channels responsible for the delayed hyperpolarisation.     

The Effect of the Lipogenic Inhibitor Cerulenin, on Macrostomal Cell Formation in Tetrahymena vorax

ABSTRACT. Macrostomal cell formation is blocked by the antibiotic cerulenin at levels of 15 μg/ml or higher. Inhibition can be reversed up to 4 h following cerulenin addition by washing and resuspending cells in new, noncerulenin-treated transforming principle. In these latter cases, additional time equal to the time spent in the inhibitor, is needed for cells to reach control values of transformation. Neither the addition of saturated or unsaturated fatty acids, cholesterol added alone or in combination with stearic acid, nor a mixture of lipids extracted from Tetrahymena vorax reversed the cerulenin effect. Radioisotope incorporation data showed while protein synthesis was reduced by the end of 1 h and tetrahymanol synthesis by the end of 2 h, little or no effect of this inhibitor occurred on RNA or fatty acid synthesis during these times. One interpretation of these results is that cerulenin, by preventing first protein synthesis and later tetrahymanol synthesis, interferes with synthesis and formation of membranes required for the microstome to macrostome transition.     

Food Vacuoles During Heat-Shock-Induced Transformation from the Microstomal to the Macrostomal Cell Type of Tetrahymena vorax1

ABSTRACT. The heat-shock method for induction of the macrostomal form of Tetrahymena vorax involves the transfer of cells to reduced nutrient medium and the application of a series of elevated temperature shocks followed by washing the protozoa into inorganic medium. The component of the procedure that had the greatest effect on food vacuoles was the heat shocks. At the end of the heat shocks, cells formed vacuoles at a lower rate than non-heat-shocked cells, but the size of the vacuoles formed was larger and the protozoa contained an increased number of vacuoles and total vacuolar membrane. The rate was further reduced by washing cells into nonnutrient medium. In the absence of the heat shocks, the medium had little effect on the capacity of the cells to form vacuoles although after 7.5 h in inorganic medium, the vacuoles formed were smaller and the protozoa possessed fewer vacuoles and therefore less vacuolar membrane. The amount of membrane required to form the cytopharyngeal pouch of the macrostomal cell type was equivalent to the surface area of food vacuoles present in cells prior to the onset of the heat shocks, but the number and surface area of vacuoles decline between the time of oral resorption and pouch development.     

Temporal pattern of arterial CO2 partial pressure during exercise in humans

The major objective of this study was to test the hypothesis that arterial CO2 partial pressure (PaCO2) does not change in transitions from rest to steady-state exercise and between two levels of exercise. Nine young adults exercised on a treadmill or a bicycle (sit or supine) for 5 min at a mild work load (heart rate = 90 beats X min-1) and then 3 min at a moderate work load (heart rate = 150 beats X min-1). In some studies the moderate work load preceded the mild work load. Arterial blood was sampled from a catheterized artery. During all exercise tasks isocapnia was not strictly maintained (F greater than 4.0, P less than 0.001). For example, a 1-to 2-Torr hypocapnia was the dominant trend during the first 15-45 s after increasing treadmill speed, and a transient hypercapnia was most prevalent when treadmill speed was decreased. During steady-state exercise PaCO2 did not deviate by more than 1-3 Torr from PaCO2 during any resting posture, and PaCO2 differences between exercise intensities and conditions did not exceed 1-2 Torr. A mouthpiece-breathing valve system was not used in most studies, but when this system was used, it did not consistently affect exercise PaCO2. Increasing inspired O2 to 40% likewise did not consistently alter exercise PaCO2. Failure to maintain isocapnia throughout exercise indicates that the matching of alveolar ventilation (VA) to lung CO2 delivery is not exquisitely precise. Accordingly it is inappropriate to base theories of the exercise hyperpnea on the heretofore contention of precise matching.(ABSTRACT TRUNCATED AT 250 WORDS)