Growth Of Tetrahymena Research Articles

COMPARISON OF PROTOZOAN AND CONVENTIONAL METHODS OF VITAMIN ANALYSIS

ABSTRACTData obtained from commonly used methods of assaying for riboflavin, vitamin B6, niacin, pantothenic acid, thiamine, biotin and vitamm B1 2 were compared to data obtained from vitamin analysis procedures employing protozoa as the test organisms. The protozoa were of the genera Tetrahymena and Ochromonas. Generally, the procedures employing Tetrahymena indicated higher levels of the vitamins in the foods analyzed, while the procedures employing Ochromonas indicated lower vitamin levels than the corresponding commonly used methods of vitamin analysis. Both protozoan and the commonly used methods for analysis of vitamins indicated higher levels of vitamins in food samples when the sample extracts received enzymatic as well as acid hydrolysis treatments. Neutralization salts formed during vitamin extraction procedures were found to interfere with both the conventional and the protozoan methods. High‐acid foods (e.g., orange and tomato juices) were found to inhibit the growth of Tetrahymena. Further studies are required to develop optimal methods of vitamin extraction and analysis in order to quantify the biologically available vitamin contents of foods.

Membrane changes during growth of Tetrahymena in the presence of ethanol.

After a brief lag period for acclimation, Tetrahymena pyriformis, strain NT-1, is capable of growing in culture medium containing high levels of ethanol. When grown in a medium having 1.6% ethanol, the membrane phospholipid composition was significantly different from that of control cells. The principal changes included a reduction in hexadecenoic acids (16 : 1 and 16 : 2) from 23% to 5% and an increase in linoleic acid (18 : 2) from 14% to 25% Similar but less pronounced changes were observed in cells grown in lower ethanol concentrations. There was also a decrease in 2-aminoethylphosphonolipid in the ethanol-grown cells from 16% of the lipid phosphorus to 6% and a coincident rise in the phosphatidylethanolamine from 39% to 46%. The lipid pattern quickly reverted to normal when ethanol was removed. In order to ascertain the effects of ethanol on membrane physical properties, freeze-fracture electron microsocopy and fluorescence polarization studies were performed. Ethanol, as expected, had a clearly detectable fluidizing influence when present at 1.6%. However, changes induced in the membrane lipids by growth in the ethanol-containing medium led to a further fluidizing effect, whether they were tested in the presence or absence of ethanol. The alterations found here were qualitatively similar but quantitatively much more pronounced than those observed in mammals chronically exposed to ethanol.

Cooperativity of hormones and hormone-like materials in Tetrahymen

1. 1. Triiodothyronine, serotonin and gramine equally enhanced the growth of Tetrahymena at 10 −9 M concentration, but equally depressed it at 10 −5 M. 2. 2. Paired combinations of the hormones showed either positive or negative cooperation, as assessed from their influence on Tetrahymena growth. 3. 3. The chemically different triiodothyronine and gramin enhanced the effect of one another, while chemically related serotonin and gramine mutually depressed it. 4. 4. The cooperative interaction of serotonin and T 3 also was negative.

EVALUATION OF PROTEIN QUALITY OF FIVE VARIETIES OF LENTILS USING Tetrahymena pyriformis W

ABSTRACTThe protozoan Tetrahymena pyriformis W was used to assess protein quality in two cultivars and three experimental lines of lentils. Cooked, freeze‐dried lentil powders and two protein fractions (legumin albumin) were evaluated and showed small but significant (P < 0.05) differences. The protein in whole (nonfractionated) lentil powder supported 15–19% of the growth supported by the control protein, casein; two of the experimental breeding lines supported more Tetrahymena growth than did the cultivars. The albumin fractions supported almost three times more growth and the legumins supported slightly growth than did the lentil powders. Supplementation studies with intact lentil powders showed sulfur‐containing amino acids to be first limiting. Lentil powders with essential amino acids added to simulate casein produced less than half as much growth as did casein.

EFFECT OF FREE AND BOUND CHLOROGENIC ACID ON THE IN VITRO PROTEIN DIGESTIBILITY AND TETRAHYMENA BASED PER OF A CASEIN MODEL SYSTEM

ABSTRACTThe effect of free and bound chlorogenic acid on the in vitro proteolytic digestion and subsequent growth of Tetrahymena pyriformis W on casein was observed. Bound chlorogenic acid caused a small, but real drop, in in vitro protein digestibility of the casein complex, and a significant inhibition in Tetrahymena growth with a resultant low Tetrahymena based PER. Free chlorogenic acid, when added to casein, did not affect its protein digestibility or its Tetrahymena based PER. Because of the severe effect bound chlorogenic acid has on the growth of Tetrahymena, caution should be exercised in the use of this organism in bioassays to measure the protein quality of proteins suspected to contain bound chlorogenic acid.

An improved method for the microbiological assay of available amino acids in proteins using Tetrahymena pyriformis

1. A standard microbiological procedure for the assay of available amino acids in proteins using Tetrahymena pyriformis has been modified by evaluating cell growth from the tetrahymanol content of the cultures by gas-liquid chromatography (GLC). 2. This modification improves the precision of the method compared with cell-counting techniques and provided automatic injection facilities are available for the GLC, the procedure is not excessively time-consuming. 3. It has been confirmed that enzymic predigestion is essential for all except the most soluble proteins and a satisfactory method using pronase has been incorporated into the microbiological procedure. 4. After enzymic digestion of the sample, growth of Tetrahymena with many proteins can be measured turbidimetrically, but particulate matter still persists with a proportion of samples, particularly those of plant origin, and the GLC method is therefore more widely applicable. It is also more precise. 5. Good agreement has been obtained between a chick bioassay and the modified Tetrahymena assay for available lysine in eight different proteins, including heat-damaged fish meals. 6. The modified procedure appeared to give good results for available tryptophan and methionine. 7. When cystine was omitted from the standard culture medium it was possible to test for available methionine plus cystine, but further work is required to assess the reliability of this particular assay.

A method for 13C enrichment of phospholipid acyl chains using Tetrahymena

A method has been devised for obtaining highly 13C enriched (20–25%) phospholipids and membranes from the ciliate Tetrahymena, the enrichment occurring in the acyl chains. This method provides both phosphatidylcholine and phosphatidylethanolamine with enriched chains. The central feature of the method is the monoaxenic growth of Tetrahymena on an E. coli mutant which has a high incorporation of exogenous acetate into fatty acid chains. The 13C NMR spectra of the phospholipids are sufficiently enriched to permit NMR relaxation studies.

Nucleocytoplasmic ratio requirements for the initiation of DNA replication and fission in Tetrahymena.

Hydroxyurea (10 mM) arrests the exponential growth of Tetrahymena by blocking DNA replication during S-phase. After removal of the hydroxyurea (HU), they have a long recovery period during which they are active in DNA synthesis. 3H-TdR uptake showed that on completion of the recovery period, the cells divide (recovery division) and enter a cell cycle which lacks G1. The frequency, size and DNA content of the extranuclear chromatin bodies (ECB) formed at this division are all markedly increased (2-4) over the corresponding values obtained from exponential growth phase controls. Microspectrophotometric analysis of macronuclear DNA content (N) coupled with the cytoplasmic dry mass (C) values suggest that specific N to C ratios (N/C) are required for the initiation of DNA replication and fission: during a normal (exponential growth) cell cycle, both N and C double, but asynchronously, so that the N/C of both post-fission-daughter cells and pre-fission cells is identical (standardized to N/C = 1) but late G1 cells have a low N/C. During a 10 hr exposure to HU, the N remains essentially the same whereas the C increases. When the HU is removed, the N increases by 4X and the C continues to increase until just prior to recovery division when it also reaches a value 4X that of the original daughter cells. Thus, the N/C = 1 is re-established. The enlarged ECB formed during recovery division may function to lower the N/C in the daughter cells, which in turn may in some way stimulate immediate DNA replication, thus eliminating G1. The elimination of G1 (and shortening in a few subsequent cell cycles) allows less time for cytoplasmic growth and results in the return of the cells to the generation time and the N and C values observed prior to the HU treatment.

Chapter 17 Manipulations with Tetrahymena pyriformis on Solid Medium

This chapter discusses the two methods for growing and cloning Tetrahymena on solid medium on petri plates. The first method involves the use of Sephadex beads on the plates to aid in restricting the movement of cells while still allowing them to remain motile and divide. The Sephadex beads on the plates aid in restricting the movement of cells while still allowing them to remain motile and divide. Most of the liquid in which cells are added to the plate is taken up by the agar and by the Sephadex, and spaces between Sephadex beads remain dry. Thus Tetrahymena cannot migrate between Sephadex beads and single cells are localized around beads in a liquid environment with sufficient nutrients to allow for motility, growth, and division. The second method involves growth of Tetrahymena in micro-holes punched in the surface of agar medium. Cells can be grown, cloned, and replica plated using this method. Thus, these methods should be very valuable in some of the steps in mutant isolation and genetic manipulation in Tetrahymena.

Changes in microbody-marker enzymes during growth of Tetrahymena pyriformis E

Marked differences in the rates of synthesis of several microbody (peroxisome) enzymes have been observed during the growth of Tetrahymena pyriformis (strain E) in shaking or static peptone medium. The specific activities of catalase and NADP + -isocitrate dehydrogenase, and of the mitochondrial enzyme fumarase, remained virtually constant during exponential growth under all conditions examined, whereas that of glycollate oxidase decreased dramatically in the course of growth. In contrast, isocitrate lyase and malate synthase were synthesized at increasing rates during growth on peptone medium with or without added acetate; highest specific activities were reached in late exponential to early stationary phase. The synthesis of these two enzymes, which appears to be coordinately regulated, was repressed in medium supplemented with glucose. Despite these variations in specific activity, glycollate oxidase as well as the key enzymes of the glyoxylate cycle remained associated with microbodies under all growth conditions tested.

Antiprotozoal Activity of Rubratoxin B

Growth of Tetrahymena pyriformis HSM was inhibited by a concentration of 25 mug of rubratoxin B per ml of growth medium. The inhibitory effect of aflatoxin B(1)-rubratoxin B mixtures on growth of T. pyriformis was induced by rubratoxin B only. Total cellular deoxyribonucleic acid increased in the presence of 5 mug of rubratoxin B per ml of growth medium, whereas higher concentrations (25 mug/ml of growth medium) were required before a decrease in total protein was observed.

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media

Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen.

A technique for cultivation of Tetrahymena in rotating bottles

A method for cultivating Tetrahymena in rotating glass bottles is described. The advantages of the rotating cultures are: 1. 1. Oxygen may be supplied in sufficient amounts for dense cultures. 2. 2. The oxygen concentration may be maintained at a predetermined level. 3. 3. Exponential growth generally continues up to about 10 6 cells/ml. 4. 4. In cultures synchronized by heat shocks, it is possible to obtain synchronous divisions at population densities up to about 2 × 10 5 cells/ml prior to the first synchronous division. 5. 5. Cultures are grown in standard glass bottles, and the volume of cultures may be varied from a few ml to 51. 6. 6. The number of cells in duplicate samples is generally reproducible within 2–3%. 7. 7. Changes in respiratory rates may be followed by an oxygen electrode. In addition some data on the growth of Tetrahymena in two different media (proteose peptoneliver extract and proteose peptone-yeast extract) are presented.

Metabolism of α-glyceryl ethers by Tetrahymena pyriformis II. Properties of a cleavage system in vitro

The biocleavage of glyceryl ethers (especially of chimyl alcohol) in Tetrahymena pyriformis W cell-free preparations was investigated. Tetrahymena crude homogenates or subcellular fractions are 45–70 times more active than has been reported for comparable rat liver preparations. The system is similar to cell-free liver preparations in that the reaction requires molecular O 2, NAD + and an NADPH-generating system as cofactors for maximal activity. However, it was shown that neither a pteridine nor any other low-molecular weight compound soluble in the microsomal supernatant is involved in this reaction. Instead, the enzyme activity is distributed as an independent entity during subcellar fractionation, being principally recovered in the pellet resulting from centrifugation of the microsomal supernatant at 250000 × g. The major product of the glyceryl-ether breakdown was identified as fatty acid produced from the hydrocarbon side chain of the chimyl-alcohol molecule. Two other products were detected, one migrating as fatty aldehyde on thin-layer chromatography and another as fatty alcohol. The formation of the aldehyde-like compound does not seem to precede the formation of the product labeled as “fatty alcohol”, and no particular accumulation of radioactive fatty aldehyde was observed by omitting NAD + from the reaction mixture or by including in it a bulk quantity of unlabeled palmitaldehyde. It was demonstrated that this ability of Tetrahymena to degrade glyceryl ethers in an in vitro system is not inducible and no significant changes in this enzyme specific activity were observed at various stages of Tetrahymena growth. Finally, the present system was found to possess comparable ability to cleave glyceryl-vinylic and glycol ethers, but not glyceryl-ether phosphatides.

EFFECT OF COMPOUNDS AFFECTING THE ADRENERGIC MECHANISM ON CELL GROWTH AND DIVISION OF TETRAHYMENA PYRIFORMIS W

Janakidevi et al. (1) showed that the ciliated protozoan, Tetrahymena pyriformis, could biosynthesize both of adrenaline and noradrenaline, while the flagellated protozoan, Crithidia fasciculata, biosynthesized only adrenaline. Accordingly, Blum et al. (2) studied the effect of reserpine on the protozoan and found that it inhibited growth and decreased the catecholamine content. Furthermore, Blum (3) investigated the effects of drugs which interacted with the action of catecholamine in mammals, on the glycogen content and growth of Tetrahymena, and suggested a relationship between glycogen synthesis and the control system for adrenergic metabolism in Tetrahymena. Later, serotonin was also discovered in both Crithidia and Tetrahymena by Janakidevi et al. (4). Furthermore, Blum et al. (5) suggested that clofibrate, a hypolipidemic agent, inhibited glyconeogenesis or activated glycogenolysis in Tetrahymena. And recently, Blum (6) reported that adrenergic control system of Tetrahymena influenced a peroxisomal enzyme which acted the first step of glyconeogenesis from acetyl-CoA. In an earlier publication from our laboratory (7), we recognized that the growth of Tetrahymena was promoted by dibenamine or ergotamine. The present paper is on the effect of the compounds used in previous experiments, on growth during 72 hours, and on the cell growth and division in one life cycle of the protozoan.

Untersuchungen über die Phosphorsäureester-Resistenz und über den Mechanismus der Entwicklung dieser Resistenz bei Tetrahymena pyriformis Gl.

The effect of 0,0-Diethyl-O-p-nitrophosphate (Paraoxon) on the growth of Tetrahymena in culture was investigated. At all concentrations tested (dilutions 1: 10.000 to 1 million) growth was depressed. After pretreatment with Paraoxon at a dilution of 1:100.000, resistence to the compound developed. Thus 5 days pretreatment increased the survival time in high concentrations of Paraoxon threefold, and 14 days ninefold, when compared with controls. Resistant and non-resistant cells were compared. After 8 days pretreatment oxygen consumption and catalase activity doubled. Acid phosphatase activity doubled following 4 weeks pretreatment and a twofold increase in cholinesterase activity was seen after 8 weeks pretreatment. No specific esterases capable of splitting phosphate esters could be shown in resistant cells. It may be significant that in resistant cells enzymes sensitive to Paraoxon are increased.

Interaction of acetate, glucose and growth conditions on glyconeogenesis and isocitrate lyase activity in tetrahymena

1. 1. Incubation of shaken cultures for 3 hr with acetate had little effect on the level of isocitrate lyase or the capacity for glyconeogenesis. 2. 2. Maintenance of cultures under static conditions for the same period led to an eight to tenfold increase in the enzyme and to a greatly enhanced ability to convert acetate to glycogen. 3. 3. Glucose prevented most of the increase in enzyme and led to a complete loss of the capacity for glyconeogenesis. 4. 4. Starved cells from non-shaken suspensions developed an increased capacity for glyconeogenesis, and the presence of acetate further increased this capacity. 5. 5. Cells from suspensions shaken with acetate had a much lower capacity. 6. 6. It is concluded that in Tetrahymena growth conditions are much more important than acetate in the regulation of glyconeogenesis and the synthesis of isocitrate lyase.

Temperature adaptation of the growth and division process of Tetrahymena pyriformis: II. Relationship between cell growth and cell replication

Replication rates and volume distributions of Tetrahymena pyriformis GL determined with a commercially available electronic cell counter are reported for several temperatures. Tetrahymena pyriformis GL was cultured in proteose peptone yeast extract medium at 28.0, 30.8, 32.5, and 33.9 °C. During the period of exponential cell replication at all temperatures, the volume distribution was constant, lognormal and the standard deviation from the median volume was temperature independent. This indicates a steady state of balanced growth at 28.0, 30.8, and 32.5 °C during exponential cell replication. It is demonstrated that the knowledge of volume distribution and generation time is insufficient for calculating rates of cell growth between divisions unless the parameters for the momentary volume distribution can be measured independently. Statistical evaluation of data from the literature confirms previously reported findings which indicate that growth of Tetrahymena over extended periods of the division cycle is approximately exponential and not linear. The previous proposition that control mechanisms for growth of Tetrahymena and Paramecium are different, is thus unnecessary. A new model, which describes the control of cell growth and cell replication is suggested. The model accounts for the lognormal distributions of generation times and volumes as well as the nearly exponential rate of growth between divisions. The mean rate of cell replication which decreases with increasing temperatures above the optimum rather than all temperatures shows a negative correlation with the median log volume of the culture. This is in accordance with the predictions of the proposed growth model and furthermore indicates that the growth rate is to a large extent independent of cell replication.

Colpidium‐Produced RNA as a Growth Stimulant for Tetrahymena*

SYNOPSIS. During logarithmic growth, Colpidium campylum produced a complex, heat‐stable material which elevated the reproductive ate of Tetrahymena subsequently inoculated into the culture. This material stimulated only early stages in the growth of Tetrahymena—effect accompanied by an increased number and size of food vacuoles. The Colpidium product was found to be RNA, precipitable from the medium by acetone. Lipids and proteins or peptides were also present in the complex; these appeared to protect the RNA from the action of chemical and physical agents.

IRON DEFICIENCY AND THE METABOLISM OF TETRAHYMENA PYRIFORMIS.

SYNOPSIS. An iron deficiency was noted for Tetrahymena grown in a proteose‐peptone‐yeast extract medium. Thus, the addition of an iron chelate and glucose was found to give consistent and luxuriant growth, whereas a number of other additives, including sodium acetate, an inorganic salt mixture lacking iron, and vitamins were found to be without influence on growth rate or cellular yield.Iron‐deficient cells showed a marked reduction of oxidative capacity when suspended in a non‐nutrient medium as well as when supplied with oxidizable substrates. A 1 hour incubation of iron‐deficient Tetrahymena with iron chelate restored oxidative capacity only when the cells were suspended in a nutrient (peptone) medium. Growth of Tetrahymena in media autoclaved with salts resulted in cells with a larger volume but with no increase in oxidative capacity.The addition of 2,4‐dinitrophenol elevated respiration of cells suspended in a non‐nutrient medium, and of iron deficient cells suspended in the growth medium, suggesting that this compound increases the availability of iron or iron containing compounds, or by‐passes the limiting steps.Respiratory quotients were lowered by an iron deficiency and elevated when the cells were grown in the presence of iron and glucose. This suggests more complete utilization of the carbohydrate, possibly due to the activity of a number of enzymes known to be iron‐containing metalloproteins that are required for oxidative glucose dissimilation.