Macrophage Activation by Tetrahymena pyriformis. I. Active Subcellular Fractions of Tetrahymena

Experiments were carried out to determine what subcellular fractions of Tetrahymena pyriformis could, after inoculation into mice, activate macrophages to kill Toxoplasma gondii in vitro. Peritoneal macrophages from mice inoculated intraperitoneally with cilia, pellicles, mitochondria, and microsomes exhibited strong toxoplasmacidal activity and had an enhanced capacity to release hydrogen peroxide (H₂O₂) by stimulation of a membrane-active agent as compared with resident macrophages. In contrast, macrophages from mice inoculated with macronuclei and postmicrosomal supernatant showed no toxoplasmacidal activity and a low level of H₂O₂ release. Similar dose response was observed on the active subcellular fractions with regard to the degree of macrophage activation. Treatment of the active subcellular fractions with heating and trypsin markedly reduced their activity.     

Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

Some Amino Acid-Folic Acid Interrelationships in Tetrahymena pyriformis

DL-serine, DL-methionine or DL-serine + DL-methionine in excess inhibited the growth of Tetrahymena pyriformis H. Excess serine was most inhibitory at high concentration of folic acid, whereas the effect of excess methionine or methionine + serine was most pronounced at low levels of folic acid. Inhibition due to excess serine was relieved by raising the level of methionine or by adding pyrimethamine to lower the effective folic acid level, and was intensified by adding Dl.-ethionine or by raising the level of folic acid. Similarly, inhibition due to excess methionine was relieved by supplying more serine or adding DL-ethionine (which reduced the amount of available methionine) and was intensified by adding pyrimethamine. Inhibition by excess methionine + serine was reversed by increasing threonine, provided there was ample guanine present. Low levels of guanine or the presence of 8-azaguanine prevented this reversal. Comparisons are made with the work of others.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.     

Macronuclear Cytology of Synchronized Tetrahymena pyriformis*

SYNOPSIS. Heat shock and stationary-phase conditions both cause fusion of nucleoli. In both cases the process is reversed when the cell is returned to normal physiological growth conditions. Fusion of nucleoli during the cell cycle of logarithmically growing cells was not observed. Likewise, fusion of nucleoli was not observed when the Padilla and Cameron(8) method of synchronization was used. The macronuclei of cells synchronized by the 1 cold-shock per cycle method(8) more closely resembled macronuclei of log-phase cells than did the macronuclei of cells synchronized by the Scherbaum and Zeuthen(12) heat-shock method.     

Toxicity of Antioxidants to Tetrahymena pyriformis*

SYNOPSIS. The cytotoxicity of 97 antioxidants of various structural types was determined for Tetrahymena pyriformis grown in a peptone medium. Cytotoxicity fo Tetrahymena, generally, was positively associated with acute oral tomicoiciity to rats but not with antioxidant pdency as measured by the Tetrahymena photodynamic assay for antioxidants. Some commonly used hindered-phenol, lipidsoluble anttioxidants, e.g., 2,6-di-tert.-butyl-P-cresol (BHT), had low toxicity (to Tetrahymena but poor antioxidant activity by the photodynamic test. Nmdihydroguahetic acid, with low toxicity to the rat and high Coxicity (to Tetrahymena, had very high activity in the photodynamic test.     

The Isolation and Identification of Kinetosome-Rich Fractions from Tetrahymena pyriformis

SYNOPSIS. Axenic cultures of Tetrahymena pyriformis W were used to obtain fractions rich in kinetosomes by alcoholdigitonin extraction techniques followed by centrifugation. The morphology of the kinetosomes was examined in the electron microscope at various stages during the isolation procedure, and compared to the morphology of the in situ kinetosome. In the latter preparation the well known cartwheel structure was present, and in addition 9 electron dense dots were displayed at the end of each spoke of the cartwheel.
The prepared kinetosomes could easily be identified during the entire process, and there was no apparent change until after the digitonin step. However, with the further fractionation, alteration was found to have taken place in the interior and in the kinetosome wall. The inside appeared empty, and we were not able to find 9 triplets in the wall but only doublets.
In view of the morphological alterations in the kinetosomerich fractions, chemical analysis still appears to us to be premature.     

Free Amino Acids in Serine-Antagonized Cells of Tetrahymena pyriformis.

Wragg, June B. (Agricultural Research Service, Beltsville, Md.), Howard Reynolds, and Michael J. Pelczar, Jr. Free amino acids in serine-antagonized cells of Tetrahymena pyriformis. J. Bacteriol. 90:748-754. 1965.-Growth inhibition of Tetrahymena pyriformis by l-serine in a chemically defined medium was reversed by l-arginine in a manner which resembled competitive antagonism. Composition of the free amino acid pools from cells grown in either a balanced amino acid mixture or a mixture with serine concentrations which inhibited growth suggested an antagonism by serine with energy-yielding reactions. Growth in media with excess serine resulted in the accumulation of higher concentrations of free cellular amino acids and an apparent increase in the rate of conversion of arginine to ornithine, as compared with growth in the balanced medium. The results suggested that serine or a metabolic product of serine interferes with the formation of pyruvic acid. In the presence of high levels of serine, arginine appeared to be metabolized more rapidly and to be spared when alanine, aspartic acid, or glutamic acid was added to the unbalanced medium.     

Electrophoretic Characterization of Classical Tetrahymena pyriformis Strains*

The electrophoretic mobility patterns of 8 enzymes have been examined in 43 classical strains of Tetrahymena pyriformis. The strains may be assorted into sets on the basis of a high degree of similarity of their mobility patterns. Strains of similar designation are frequently in different sets, whereas differently labeled strains may be in the same set. It is proposed that new strain designations be made on the basis of phenotypic similarity.     

An Apparent Carbohydrate-Amino Acid Interaction in Tetrahymena pyriformis

SYNOPSIS. Experiments were designed to examine the nature of the response of Tetrahymena pyriformis to inhibitory levels of L-serine when growing in defined media with carbohydrate as mono-, or polysaccharide. Growth curves indicated that, when a polysaccharide is being utilized, inhibition by serine affects at least 2 different loci. At one locus, inhibition causes a marked increase in the growth lag, at the other a significant decrease in the maximum rate of growth. Since the inhibition was completely reversible by glucose supplementation, it is evident that the initial inhibition, expressed as increased lag time, is specifically related to uptake or metabolism of the polysaccharide, i.e., it precedes glucose in the metabolic sequence. The inhibition expressed as decreased rate of maximum growth appears to be due to interference at some stage in the glycolytic pathway. The results suggest that in metabolizing a polysaccharide, T. pyriformis utilizes 2 mechanisms simultaneously. One involves phagocytic ingestion of molecular dextrin followed by phosphorylytic cleavage whereby the energy of the glycosidic bond is utilized to form glucose-1-phosphate. The 2nd mechanism depends upon elaboration of extracellular hydrolytic enzymes to cleave the polysaccharide to glucose, which enters the glycolytic pathway via glucose-6-phosphate in the usual manner.     

Further Electrophoretic Characterization of Strains of Tetrahymena pyriformis*

Mobility patterns of 5 isozymes in strains of Tetrahymena pyriformis were demonstrated using polyacrylamide disc gel electrophoresis. Six stock strains were compared in these patterns to 4 strains representative of each of the previously described 4 major “phenotypic sets.” Stock strains segregated into predicted “phenosets,” and essentially confirmed validity and reproducibility of such a discrimination method. The proposal that new strain designations be assigned on a basis of “phenoset” conformity is reaffirmed.     

The Conversion of Phenylalanine to Tyrosine in Tetrahymena pyriformis*

SYNOPSIS. Phenylalanine hydroxylase could not be assayed in extracts of Tetrahymena pyriformis strain W in a system by which the enzyme could be assayed in rat liver extracts. Isotopically labelled phenylalanine, however, was converted to tyrosine by growing or washed cells. Growth conditions which allowed limited synthesis of unconjugated tetrahydropteridine severely reduced the ability of the cells to synthesize tyrosine from phenylalanine. The presence of glucose and acetate in the growth medium resulted in elevated free tyrosine pools and an increased capacity of washed cell suspensions to convert phenylalanine to tyrosine. It would appear that the putative phenylalanine hydroxylation system is not subject to the repressive effects of glucose and acetate which apply to the enzymes of tyrosine catabolism. The significance of this distinction is discussed.     

Activation of Glycogen Phosphorylase by Cyclic AMP in Tetrahymena pyriformis1

Glycogen phosphorylase in Tetrahymena pyriformis was activated by a Mg²⁺ ATP-dependent process and this activation was further increased by the addition of cyclic AMP. When the enzyme activity in subcellular fractions was measured, it was largely associated with the glycogen fraction but was no longer activated by ATP and cyclic AMP. Mixing the glycogen fraction and cytosol fraction together restored the effects of ATP and cyclic AMP on phosphorylase activity. These findings suggest that glycogen phosphorylase associated with Tetrahymena glycogen granules may be regulated by cytosolic factor(s) with cyclic AMP.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

An Ultrastructural Study of Ingestion and Digestion in Tetrahymena pyriformis*

SYNOPSIS. When the structures involved in digestive events in T. pyriformis are examined at the electron microscope level, some information is added to that long known from light microscopy. The food trapping mechanism consists of the three membranelles, undulating membrane, oral ribs, and a “valve” apparently closing the opening to the cytopharynx. Both of the latter structures are supported by microtubules. Fibers extend internally from the cytopharynx and are closely associated with the food vacuole as it forms. Clear vacuoles resembling pinocytic vacuoles appear to arise from differentiated areas of the pellicle and plasma membrane. These vacuoles may fuse with primary lysosomes. Hydrolases are thus contributed to the pinocytic vacuoles which may then fuse with food vacuoles. When first formed food vacuoles contain no hydrolases but may acquire them directly, from primary lysosomes or from pinocytic vacuoles. Digestion proceeds to completion in the food vacuole, at which time soluble food products are released to the cytoplasm. Undigested materials are lost through the cytopyge. In stationary growth phase cells autophagic vacuoles form containing mitochondria and other cellular particulates. Such vacuoles probably contain hydrolases when formed and they may receive others by fusion with primary lysosomes.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Survival of Tetrahymena pyriformis and Paramecium aurelia Following Freezing*

SYNOPSIS. Tetrahymena pyriformis strains E, A-136 31C and IMT II survived freezing in 10% dimethylsulfoxide when the temperature was lowered to freezing at 4.5 C/min. Survival was then obtained for at least 128 days by lowering the temperature rapidly to 95°C. Of the 3 strains, T. pyriformis IMT II was most resistant to the effects of freezing. Its volume averaged about half that of either of the other strains and may have contributed to the differences in survival. In addition to differences among strains, a medium relatively low in the concentration of nutrients, a culture nearing peak population, and a rate of cooling of 4.5 C/min, all gave best survival. Paramecium aurelia regained motility after being frozen in 6 to 7.5% dimethylsulfoxide for as long as 7 days at either –27 or –196 C, but cultures were obtained only after storage for 20 min at –27 C. A concentration of 6 to 7.5% dimethyl-sulfoxide, cooling at 4.5 C/min, and culture media containing Aerobacter aerogenes or composed of a commercially available composition were all required for survival of P. aurelia.     

Temperature Effects on Maturity Periods in Tetrahymena pyriformis Syngen 1*

Cells of Tetrahymena pyriformis syngen 1 grown at 30 C after conjugation achieve sexual maturity more quickly than do cells grown at 19 C, whether time is measured in numbers of cell divisions or in terms of absolute time. This result is achieved regardless of the temperature at which conjugation and nuclear reorganization occur. These observations differ from those of other workers investigating Paramecium, and suggest that the long term “chronometer” is more tightly coupled to cell division in Paramecium multimicronucleatum and Paramecium caudatum than in Tetrahymena pyriformis.