Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

Outgrowth and sporulation studies on Clostridium botulinum type E: influence of isoleucine.

A defined medium (CDM) is described which supported growth and sporulation of type E strains of Clostridium botulinum, but not sporulation of other serotypes of C. botulinum or C. sporogenes. As compared to growth in complex medium, spore outgrowth was delayed and both the growth rate and the cell yield was reduced. However, efficiency of sporulation of the type E MSpt strain in a chemically defined medium (CDM) was the same as that in complex medium and, in fact, sporulation was nearly synchronous and completed within 3 h of the first appearance of phase-bright endospores, compared with completion in 9 h in TPGY. Growth studies with CDM, from which single amino acids were omitted, showed that isoleucine was essential for outgrowth of heat-activated spores of the MSp+ strain, whereas valine was required for that of the Ts-25 mutant. Radioactive isoleucine was incorporated by germinating MSp+ spores at an earlier stage and at a more rapid rate than labelled methionine or mixed amino acids. Uptake studies showed that isoleucine accumulated in a prominent acid-soluble pool during outgrowth, a period when its incorporation into protein was not evident. The results suggest that the isoleucine may be required for a purpose other than protein synthesis during outgrowth.     

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 (10⁻¹⁰ M) to cells in CDM prevents the surface mediated death. Since detergents/surfactants like SDS (7 × 10⁻⁵ M), NP-40 (2 × 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. © 1996 Wiley-Liss, Inc.     

Starved Tetrahymena thermophila cells that are unable to mount an effective heat shock response selectively degrade their rRNA.

Tetrahymena thermophila cells that had been shifted from log growth to a non-nutrient medium (60 mM Tris) were unable, during the first few hours of starvation, to mount a successful heat shock response and were killed by what should normally have been a nonlethal heat shock. An examination of the protein synthetic response of these short-starved cells during heat shock revealed that whereas they were able to initiate the synthesis of heat shock proteins, it was at a much reduced rate relative to controls and they quickly lost all capacity to synthesize any proteins. Certain pretreatments of cells, including a prior heat shock, abolished the heat shock inviability of these starved cells. Also, if cells were transferred to 10 mM Tris rather than 60 mM Tris, they were not killed by the same heat treatment. We found no abnormalities in either heat shock or non-heat shock mRNA metabolism in starved cells unable to survive a sublethal heat shock when compared with the response of those cells which can survive such a treatment. However, selective rRNA degradation occurred in the nonsurviving cells during the heat shock and this presumably accounted for their inviability. A prior heat shock administered to growing cells not only immunized them against the lethality of a heat shock while starved, but also prevented rRNA degradation from occurring.     

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.     

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.     

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Presence (hPL, prostaglandin) and absence (triiodothyronine, thyroxine) of hormones in Tetrahymena: experimental facts and open questions

The RIA technique detected prostaglandin (PGF2) and human placetal lactogen (hPL) in Tetrahymena cultures grown in bacto tryptone + yeast extract medium which, however, itself contained these hormones. About one to two per cent of the total hormone content of the medium was demonstrated intracellularly. Treatment with diiodotyrosine (T2), which is known to stimulate the growth of Tetrahymena, was followed by a decrease in the intracellular prostaglandin level. Triiodothyronine and thyroxine were not detected in Tetrahymena or in the medium, and did not appear in it on induction with TSH either. In the light of these observations it might well be doubted that prostaglandin was native in Tetrahymena: the use of synthetic media, and/or a reliable demonstration of the hormone content of the growth medium is recommended for evidence of hormone biosynthesis by unicellular organisms.     

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.     

Signalling in cell growth and death: adequate nutrition alone may not be sufficient for ciliates

The initial inoculum level of Tetrahymena in a chemically defined medium determines whether the cells are capable of exponential growth. Below 750 cells ml^?1, the cells fail to go into exponential growth and will die within about 20 hours. By adding certain growth stimulants, death can be postponed and the cells begin to grow after a delay which depends on the intensity of the signal. The implication is that autocrine growth factor expression might be required for cells to grow, and that these stimulants either assist its production or lower the cell threshold to its action. The findings in Tetrahymena are reviewed, and the advantages of having a cell system in which all the components of the medium can be carefully controlled is recognised.     

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.     

Lowered resistance to lytic agents in sucrose vacuolation.

Vacuolation in fibroblasts cultivated in the presence of sucrose is associated with progressive accumulation of the undigestible sugar. In radioisotope experiments the process lasted several days, and when the cells were subcultured back into a medium devoid of sucrose the label was also lost after several days. This type of vacuolated cells is more fragile when it is challenged with lytic agents. 51Cr-labelled LS fibroblasts released more radioactivity when they had been growing in the presence of sucrose, whether they were suspended in media of decreasing osmolarity, in dilutions of various surfactants, exposed to high temperatures, or subjected to mechanical stress. It is concluded that these cells exhibit a lower resistance when exposed to unfavourable environments, but retain their viability in growth media despite some morphological and biochemical alterations.     

Adverse impact of heat stress on embryo production: causes and strategies for mitigation

The production of embryos by superovulation is often reduced in periods of heat stress. The associated reduction in the number of transferable embryos is due to reduced superovulatory response, lower fertilization rate, and reduced embryo quality. There are also reports that success of in vitro fertilization procedures is reduced during warm periods of the year. Heat stress can compromise the reproductive events required for embryo production by decreasing expression of estrus behavior, altering follicular development, compromising oocyte competence, and inhibiting embryonic development. While preventing effects of heat stress can be difficult, several strategies exist to improve embryo production during heat stress. Among these strategies are changing animal housing to reduce the magnitude of heat stress, utilization of cows with increased resistance to heat stress (i.e., cows with lower milk yield or from thermally-adapted breeds), and manipulation of physiological and cellular function to overcome deleterious consequences of heat stress. Effects of heat stress on estrus behavior can be mitigated by use of estrus detection aids or utilization of ovulation synchronization treatments to allow timed embryo transfer. There is some evidence that embryonic survival can be improved by antioxidant administration and that pharmacological treatments can be developed that reduce the degree of hyperthermia experienced by cows exposed to heat stress.     

Kinesin-II Is Preferentially Targeted to Assembling Cilia and Is Required for Ciliogenesis and Normal Cytokinesis in Tetrahymena

We cloned two genes, KIN1 and KIN2, encoding kinesin-II homologues from the ciliate Tetrahymena thermophila and constructed strains lacking either KIN1 or KIN2 or both genes. Cells with a single disruption of either gene showed partly overlapping sets of defects in cell growth, motility, ciliary assembly, and thermoresistance. Deletion of both genes resulted in loss of cilia and arrests in cytokinesis. Mutant cells were unable to assemble new cilia or to maintain preexisting cilia. Double knockout cells were not viable on a standard medium but could be grown on a modified medium on which growth does not depend on phagocytosis. Double knockout cells could be rescued by transformation with a gene encoding an epitope-tagged Kin1p. In growing cells, epitope-tagged Kin1p preferentially accumulated in cilia undergoing active assembly. Kin1p was also detected in the cell body but did not show any association with the cleavage furrow. The cell division arrests observed in kinesin-II knockout cells appear to be induced by the loss of cilia and resulting cell paralysis.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Involvement of ERK and protein tyrosine phosphatase signaling pathways in EGCG-induced cyclooxygenase-2 expression in Raw 264.7 cells

The cellular stress response which can be elicited by a variety of physical or chemical stressors challenges the homeostatic mechanisms of the cells. Two stressors may interact such that, for example, in the presence of a defined thermal stress ("costress") a second weak stressor like electromagnetic fields (50 MHz, 100 microT) produces strong biological effects. Based on the apparent interaction of these stressors a concept is suggested that explains the observed effects and defines the limits of cellular homeostasis in general terms. The homeostatic potential of a cell and hence the ability to cope with stressors can be altered by eliciting or depressing the heat shock response. This manipulation has several promising medical applications.     

Induction of heat shock proteins by canavanine in Tetrahymena. No change in ATP levels measured in vivo by NMR

During induction of the heat shock response by temperature jump in the protozoan Tetrahymena, a decrease in cellular ATP levels occurs within minutes and cells become thermotolerant. Treatment of Tetrahymena with the amino acid analog canavanine also induces synthesis of heat shock proteins, but more slowly than by temperature jump. No changes in cellular ATP levels were observed during the course of canavanine induction of heat shock protein synthesis measured in vivo by the technique of 31P NMR spectroscopy. Tetrahymena do not become thermotolerant following induction of heat shock protein synthesis with canavanine. However, Tetrahymena will develop thermotolerance in the presence of canavanine if they are first subjected to a nonlethal temperature jump before exposure to a normally lethal temperature.     

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.