Effects of Pluronic F-68 on Tetrahymena cells: protection against chemical and physical stress and prolongation of survival under toxic conditions.

The effects of the non-ionic surfactant Pluronic F-68 (0.01% w/v) on Tetrahymena cells have been studied. A marked protection against chemical and physical stress was observed. The chemical stress effects were studied in cells suspended in buffer (starvation) or in buffers with added ingredients from a chemically defined medium (Ca2+, Mg2+, Na+, K+, trace metal ions). The physical stress was due to mechanical stress or hyperthermia. The data show that Pluronic: (a) prolongs the survival of low concentration cell suspensions during starvation; (b) prevents the cell death caused by low concentrations of Ca2+ (70 microM); (c) prolongs the survival of cells exposed to higher ion concentrations (10 mM Ca2+, or Na+ or K+); (d) postpones the death caused by trace metal ions like Zn2+, Fe3+ and, Cu2+; (e) protects cells from the death caused by shearing forces; and (f) prolongs the survival of cells exposed to hyperthermia (43 degrees C). The cellular survival is increased at reduced temperatures (e.g. 4 degrees C instead of 36 degrees C) and at increased cellular concentrations (e.g. 100 cells ml(-1) instead of 25 or 10 cells ml(-1)). There is no effect of pre-incubation with Pluronic. The protective effect of Pluronic towards Tetrahymena is observed for concentrations in the range from 0.001 to 0.1% w/v.

Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition.

We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca2+ (68 microM) and Mg2+ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca2+ and Mg2+ can be abolished by the addition of insulin (10(-6) M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.

Expression of Tetrahymena snRNA gene variants including a U1 gene with mutations in the 5' splice site recognition sequence.

The expression of U1, U2 and U5 snRNA gene variants has been studied under different physiological states of Tetrahymena. Variants of all three snRNA genes are expressed. Among the snRNAs detected is U1-3, a variant with 66 mutations compared to the normal U1 snRNA. Three of these mutations affect the 5' splice site recognition sequence. The U1-3 snRNA is present in a few hundred copies per cell. The expression of Tetrahymena snRNA genes is independent of the physiological state of the cell.

Surface mediated death of unconditioned Tetrahymena cells: effect of physical parameters, growth factors, hormones, and surfactants.

A new form of cell death has been observed. The death occurs at liquid-air interfaces when Tetrahymena cells are grown in a chemically defined medium (CDM) at low inocula. The cells die by lysis at the liquid-air interface (medium surface), which they reach due to negative gravitaxis as well as positive aerotaxis. When the cells are grown in a closed compartment, with no liquid-air interface, the death is not observed, and the cells proliferate. Cloning of cells in CDM is thus possible. The addition of effectors such as NGF (10(-11) M), EGF (10(-10) M), PDGF (10(-10) M), and insulin (10(-7) M) to cells in CDM prevents the surface mediated death. Since detergents/surfactants like SDS (7 x 10(-5) M), NP-40 (2 x 10(-5) M), Tween 80 (10(-4))% w/v), Pluronic F-68 (10(-7) M), and the biosurfactant surfactin (10(-6) M) have the same effect, we suggest that the effectors act by stimulating the cells to exudate surfactant(s) of their own. Furthermore, lyzed cells and exudates from living cells (pre-conditioned medium) prevent the death. In conditions with liquid-air interfaces, certain physical parameters are of great importance for the survival of cells at low inocula. The parameters are the distance to the surface, the temperature, and the inoculum. By increasing the height of the medium, lowering the temperature, and increasing the inoculum of the culture, the survival can be greatly enhanced. There is no evidence for programmed cell death (PCD) or apoptosis.

Physiological parameters affecting the chemosensory response of Tetrahymena.

We have investigated the significance of a number of physiological parameters in the preparation of cells for experiments on chemokinesis in Tetrahymena. The study comprises (1) growth state of the cells, (2) composition of the starvation medium, (3) concentration of cells during starvation, (4) oxygen saturation of the starvation medium, (5) temperature during starvation, and (6) starvation period. By controlling the physiological state of the cells, we significantly improved the reproducibility of the results obtained in assays for chemokinesis in Tetrahymena. In short, cells optimal for chemokinesis at an assay temperature of 28 degrees C should be starved from the exponential growth phase in a concentration below 2 x 10(5) cells ml-1 for 10-20 h. The surface-to-volume ratio of the starvation medium--water or Hepes buffer--should be about 5 cm-1 (or more) to ensure more than 95% oxygen saturation of the starvation medium. Maximal chemosensory responses were obtained if the cells were starved at 21 degrees C. The chemokinetic potential of the cells decreased significantly, as did the levels of the ratio of ATP to ADP, if cells were starved at higher temperatures. A tentative correlation between the ATP level in the cells and the chemosensory potential of the cells has been found. We suggest that chemokinesis is a constant quality of Tetrahymena, because we found no sign that prolonged starvation or other changes applied to the cells produced an up-regulation of the chemosensory response. Apparently, starvation is obligatory only to remove the growth medium (which is itself a very potent attractant), thereby making the cells sensitive to the chemoattractants.

An improved quantitative assay for chemokinesis in Tetrahymena.

This paper presents a quantitative and sensitive assay for the measurement of chemosensory behavior in Tetrahymena. The two-phase assay is easy to perform in large quantities, so a variety of compounds can be screened under comparable conditions. A suspension of 2 x 10(5) cells ml-1 (the upper phase) is starved for 20-40 h and then gently placed on top of a 5% solution of Metrizamide (the lower phase) in a disposable microcuvette. The optical density of the lower phase is monitored at 600 nm with an automated spectrophotometer at selected time points. Optimum sensitivity of the assay is achieved when the cells slowly but continuously enter the lower phase, so that about 5% of them will be in the lower phase within 30 min. Optimal chemosensory responses occurred in Tetrahymena thermophila at about 25 degrees C. The response was delayed at 15 degrees C and markedly reduced at 35 degrees C. The data suggest three bases for quantifying the response in the assay: (1) initial slope of the absorbance versus time; (2) final maximal absorbance within the time period of measurement; and (3) signal-to-noise ratio (S/N) at a fixed time. We have quantified--in terms of S/N--the chemosensory responses in Tetrahymena for the following compounds: beta-endorphin, fibroblast growth factor, insulin, and platelet-derived growth factor (PDGF); these substances were active in nanomolar concentrations, and the maximal S/N was between 3 and 5.1. Acetylcholine was active only in millimolar concentrations; maximal S/N was 4.1 at 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Certain fatty acids and steroids protect Tetrahymena from cell division stress caused by shaking.

Cultures of Tetrahymena are routinely shaken to ensure proper access to oxygen. Recent work showed that growth of dilute cultures (inocula < 10(4) cells ml-1) of T. pyriformis was sensitive to shaking. Addition of oleic acid (9 microM) or linoleic acid (140 microM) before or at the onset of shaking gave considerable protection to the cells. A similar effect was seen with ergosterol (25 microM) and to some extent with cholesterol (100 microM). Octanoic acid (20 microM), palmitic acid (140 microM) and palmitoleic acid (100 microM) had no effect. Paraquat (230 microM), which induced peroxidation of unsaturated fatty acids, increased the effect of shaking-induced cell division stress. Such results may be due to changes in the membrane composition of Tetrahymena. It has not been possible to demonstrate differences in the 14C-oleic acid labelling of phospholipids of cells with and without shaking.

Characteristics of dividing and non-dividing Tetrahymena cells at different physiological states.

Eight defined physiological states of Tetrahymena pyriformis are described. For dividing cells the states comprise: 1. Exponentially growing cells, 2. Cells at late exponential growth phase, 3. Cells kept at a high cell concentration, 4. Cells shifted up or down by change of medium, temperature or degree of aeration. For non-dividing cells the states are: 5. Cells at stationary phase, 6. Cells during starvation, 7. Cells during shift-up after long-term starvation, 8. Cells at self-induced hypoxia. The different cellular states are described by one or more of the following characteristics: growth rate, volume, swimming speed, oxygen consumption and by the oxygen saturation and the pH in the medium. The results show that T. pyriformis grows equally well in proteose-peptone (PY) medium from 1 cell ml(-1) to 10(3) cells ml(-1) as from - e.g. - 10(2) to 10(5) cells ml(-1). The maximum cell concentration obtained depends on the medium and the availability of oxygen. At shift-down by decrease of temperature the cells grow slower and obtain a considerable oversize. Single cells tolerate starvation for 12 days. The cell volume (electronically determined) decreased from about 7000 µm(3) to about 200 µm(3). Long-term starved cells may be upshifted. Thereby growth without cell division can be studied until the cell volume approaches 2100 µm(3) which is the minimum volume of division competence. Under certain conditions cells may grow into self-induced hypoxia leading to growth arrest. These cells will attain an oversize. The swimming speed at 28°C of exponentially growing cells is 0.33 to 0.59 mm sec(-1) depending on the medium. At lower temperature the swimming speed is decreased. In PY-medium the values are: 28°C (0.57), 16°C (0.50), 9°C (0.37). During starvation the swimming speed decreases from about 0.6 to about 0.1 mm sec(-1) (after 6 days). The oxygen consumption is for state 1 cells: 3.9 µl O(2)/10(6) cells min(-1) (maximal value). The value of hypoxic cultures is 2.1, for cells kept at high concentration 0.4, and for starved cells (24 h) 0.2.

Chemosensory behaviour of Tetrahymena.

Free swimming cells of the ciliated protozoan Tetrahymena are attracted to certain chemicals by chemokinesis. However, a special type of chemotaxis in response to a chemical gradient is found in cells gliding very slowly in semisolid media. In contrast to classical chemotaxis by leukocytes, which is solely positive towards chemo-attractants, the oriented chemokinesis by gliding Tetrahymena involves both positive and negative elements. The major chemo-attractants are peptides and/or proteins, and they may be compounds which signal the presence of food in the natural environment of this freshwater phagotroph.

A paper membrane filter assay for ciliate chemoattraction.

A quantitative bioassay for ciliate chemoattraction based on the Boyden assay is described with the ciliates Tetrahymena thermophila and Tetrahymena pyriformis as test organisms. A chamber is separated into two compartments by a Whatman 3MM filter, and a suspension of starved cells (approximately 10(5) cells/ml) is placed in one compartment and a solution containing attractant in the other. The gradient of chemoattractant across the filter causes the cells to swim through the filter into the attractant-containing compartment where their appearance is determined by electronic cell counting. The assay described is convenient with a signal-to-noise ratio of approximately 10. It is shown here to work with the attractants proteose peptone and platelet-derived growth factor.

Chemotaxis in tetrahymena.

Chemotaxis - that is oriented locomotion of single cells - was shown by motion analysis of Tetrahymena thermophila. An electronic registration of swimming tracks was carried out in gradients of chemoattractant established in a modified Zigmond chamber. The attractants used were proteose peptone, platelet extract and fibroblast growth factor. From 5 to 55 minutes after addition of the chemoattractant, 65% of the cells are oriented within the 180° angle segment towards the attractant compared to 51% in the control, containing no attractant. The figures are based on the measurement of 1567 and 499 tracks, respectively. Under the conditions used, cell migration towards the attractant is caused, solely, by an oriented movement (Chemotaxis) since the swimming speed was unaffected by the presence of the attractant. Similar results were obtained using T. pyriformis cells.

Division competence in Tetrahymena: determination of minimum cell volume and rate of nutrient uptake.

Cell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased. When the cell volume increase per time per cell in a given medium is expressed as a function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 microns 3 for T. pyriformis at 28 degrees C. The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC. The increase in cell volume per unit of time for a given cell depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis-Menten-like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2.s. The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2.s) increases the ability of growth in more concentrated media.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell volume and dry weight of cultured Tetrahymena.

Cultures of Tetrahymena pyriformis, T. hegewishi and T. malaccensis have been studied with regard to control of cell volume and cellular dry weight. Cell volume was measured on cells suspended in 0.9% sodium chloride + 0.1% sodium azide using a Multisizer cell counter (Coulter). Tetrahymena were grown at different temperatures and under various up- or downshift conditions. In all cases the changes in cell volume are paralleled by changes in cellular dry weight. The volume and the dry weight of a Tetrahymena cell are determined by the particular medium and the growth temperature. Large cells are seen in concentrated media and at low growth temperatures resulting in cell volumes up to 17,000 microns 3, whereas starving cells decrease gradually towards 700 microns 3 or even smaller sizes. It is proposed that lag phase observed at up- and downshift is to a large extent due to the necessary adjustment of the cell volume to the new conditions.

Immune hemolytic assay for identification of human anti-dsDNA antibodies with DNA-coated red blood cells as target cells.

A hemolytic assay was developed with the primary aim of being able to identify human lymphocytes producing anti-dsDNA antibodies found in patients with systemic lupus erythematosus (SLE). The coating of sheep red-blood cells with DNA was performed after precoating the cells with poly-L-lysine. The DNA-SRBC were lysed by anti-DNA antibodies from SLE sera, and the percent hemolysis was found to correlate with the anti-DNA activity demonstrated by the Farr assay (r = 0.87). Single-stranded DNA at the surface of the coated cells could be removed after digestion with nuclease S1. The effect of the digestion was verified by SLE serum specific for single-stranded DNA. With slight modifications, the target cells may be used to determine not only the titer of anti-DNA antibodies but also the complement-consumption and immunoglobulin classes of the anti-DNA antibodies.

Parameters affecting the maximum cell concentration of Tetrahymena.

In cultures with efficient aeration a maximum cell concentration (MCC) of 6 X 10(5) cells/ml (defined medium) and 5.5 X 10(6) cells/ml (broth) can be reached. By culturing within Millicells with excess supply of medium and efficient removal of waste products a physical limit for MCC of about 13 X 10(6) cells/ml is reached.

Conjugation of DNA to erythrocytes.

DNA was conjugated to sheep red blood cells (SRBC) by chemical methods using CrCl3, poly-L-lysine or methylated bovine serum albumin as conjugation agents and by a physical method where conjugation was accomplished by incubation at 45 degrees C. The degree of conjugation was estimated using 32P-DNA (mean size 1 kbase pairs). Employing the CrCl3 method 5.8 +/- 3.6 micrograms DNA were conjugated per 10(8) SRBC at a concentration of 70 micrograms DNA/10(8) cells. At the same DNA concentration in the incubation medium 3.0 +/- 0.6 microgram DNA/10(8) cells were conjugated by poly-L-lysine, 4.1 +/- 0.8 microgram DNA/10(8) cells by methylated bovine serum albumin and approximately 4 micrograms DNA/10(8) cells when the cells were incubated at 45 degrees C. Cells conjugated with DNA by CrCl3 showed linearly increasing conjugation with increasing concentration of DNA. Cells conjugated by poly-L-lysine (pLL) or methylated bovine serum albumin seemed to be saturated by DNA at 30 micrograms DNA per 10(8) cells. At 45 degrees C the spontaneous adhesion of DNA to SRBC increased in the concentration range investigated. The degree of conjugation of DNA to SRBC was influenced by pH, and Ca2+.pLL-conjugated DNA-SRBC, but none of the other preparations were lysed in a hemolytic assay using anti-DNA antiserum from a patient with systemic lupus erythematosus.

Small nuclear RNAs in the ciliate Tetrahymena.

We have isolated and partially characterized a family of small nuclear RNAs (snRNAs) from three different species of the protozoan Tetrahymena. We find six distinct snRNAs ranging in size from 100 to 250 nucleotides. The two largest snRNAs, as well as an abundant, heterogenous group of smaller snRNAs are found in the nucleolar RNA fraction. None of the snRNAs are transcription products of the ribosomal RNA gene or its flanking regions, as shown by hybridization tests. The snRNAs are metabolically stable as determined by pulse/chase experiments and several of them contain a number of modified nuclotides. The snRNAs from Tetrahymena all have slightly different sizes from mammalian snRNAs. The cap structure of the snRNAs from Tetrahymena differs from that of the snRNAs from mammalian cells, but has not yet been fully characterized. The relative amount of snRNAs to total RNA is less in Tetrahymena (greater than 0.1%) than in mammalian cells (2%).

Dimers of 5 S RNA and 5.8 S RNA in low molecular weight RNA preparations.

When purified 5 S RNA is exposed to concentrated solutions of sodium chloride or sodium phosphate, it is partly converted to the 5' S conformer but also partly to dimers and oligomers of 5 S RNA which are stable during gel filtration and electrophoresis. Presence of 5 S dimers can not be demonstrated in cellular RNA preparations extracted with phenol. 5.8 S RNA dimers are observed in RNA from cells extracted with phenol at 40-60 degrees C. No 5.8 S dimer is observed when RNA is extracted at 0 degrees C. The amount of dimer increases with the temperature of extraction and may account for about 10% of the total 5.8 S RNA. When RNA extracted at 40-60 degrees C is electrophoresed on polyacrylamide gels under nondenaturing conditions the 5.8 S RNA dimer co-migrates with the low molecular weight RNA component L.