Contractility of Glycerinated Amoeba proteus and Chaos-chaos*

Immediate contact with large volumes of cold 50% (v/v) buffered glycerol preserved typical ameboid shape of Chaos chaos and Amoeba proteus with no visible distortions. These technics allowed determination of the contraction sites in these glycerinated models upon application of ATP-Ca-Mg-solutions. The ectoplasmic tube was the main site of contraction. Preliminary EM investigations revealed thick and thin filaments, associated with the ectoplasmic tube near the plasmalemma, which appeared to be the basis for the contractility of the ectoplasmic tube. There was no predominant contraction of the pseudopodial tips or the endoplasm in these models. The changes of volume were as much as 50%, and in some cases were not accompanied by any change in the length of the ameba; however, lengthwise contractions of the ectoplasmic tube in some amebae occurred to as much as 25%. The data substantiate a basic requirement of the ectoplasmic tube contraction theory of ameboid locomotion.     

Scanning Electron Microscope Observations of Amoeba proteus During Phagocytosis*

SYNOPSIS. Phagocytosing Amoeba proteus at different stages of forming foodcups have been observed by scanning electron microscopy. A nonphagocytosing ameba is characterized by dorsal and lateral ridges running longitudinally over the posterior half of the cell and its attachment to the substrate over small areas. When stimulated by prey organisms, the ameba loses polarity and ridges, and adheres to the substrate more firmly over a wider area of contact. Then it forms broad pseudopods to surround its prey and this results in the formation of foodcups. The surface of all amebae is covered with small projections, and membranous blebs are often seen on the surface of phagocytosing organisms.     

Subcellular Effects of Cytochalasin B and Dimethylsulfoxide on Paramecium aurelia*

SYNOPSIS. Paramecium aurelia syngen 4, stock 57 (sensitive) cultivated in Cerophyl infusion were exposed to cytochalasin B CB and dimethylsulfoxide (DMSO), the solvent for CB, to distinguish between the effects of these agents on a cellular system. DMSO significantly inhibited survival, fission rate, [³H]leucine incorporation, and cell size. CB-treated cells generally had slower division and poorer survival rates than cells exposed to the equivalent DMSO concentration, although the [3H]leucine incorporation was generally greater at the lower CB concentrations than for DMSO alone. As seen by electron microscopy and a new grycerination technic for observing polysomes, DMSO caused nuclear (nucleolar, chromatin) abnormalities as well as membrane degradation and polysomal breakdown; CB caused the formation of aberrant membrane structures and ribosomal tetramers, crystals, and tubes.     

Induction of Temperature Sensitivity in Paramecium aurelia by Nitrosoguanidine*

Paramecium aurelia cells were exposed to N-methyl-N-nitroso-N′-nitroguanidine for periods of 15–30 min. The lethality in homozygous clones derived from treated cells depends on the time of treatment within the cell cycle. Exposures at interfission ages 0.04, 0.40, and 0.80 were tested yielding lethalities of 12.5, 44 and 2%, respectively. These results correlate with the period of DNA synthesis in the micronuclei. A temperature sensitive mutant has been found which cannot live at 31 C but divides at ～1 fission per day at 19 and 25 C. The rise in temperature from 19–25 C does not significantly change the fission rate whereas in normal cells it would be doubled. Genetic analysis shows that this mutation is caused by a single recessive gene.     

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.     

The Axenic Culture of Strains of the Various Syngens of Paramecium aurelia*

SYNOPSIS Strains of the various syngens of Paramecium aurelia respond differently to the culture media developed for Strain 299 of syngen 8. Not only is there a variety of response between the syngens, but strains belonging to the same syngen respond differently to the 3 growth media.     

Effect of Chloramphenicol on Bacterial Endosymbiotes in a Strain of Amoeba proteus*

SYNOPSIS. The effect of chloramphenicol (CAP) on the bacterial endosymbiotes of a strain of Amoeba proteus was studied by growing the symbiotic amebae in media containing 0.5–1.6 mg/ml CAP for up to 4 weeks. Treatments with CAP caused such ultrastructural changes as expansion of the nuclear zone and deformation of symbiotes. The CAP treatment also damaged the mitochondria, e.g. disappearance of central and protrusion of peripheral cristae. Number of bacteria per ameba decreased to < 10% of control in CAP-containing media, but no viable amebae became completely free of symbiotes. The resuts supported previous studies that amebae were dependent on endosymbiotes.     

Growth of Particle-Bearing and Particle-Free Paramecium aurelia in Axenic Culture*

SYNOPSIS. Several strains of particle-bearing and particle-free Paramecium aurelia have been cultivated in an axenic medium composed of proteose peptone, trypticase, yeast nucleic acid, MgSO⁴.7H²O, TEM-4T (diacetyl tartaric acid esters of tallow monoglycerides), stigmasterol and a mixture of vitamins. The “yeast fraction,” an indispensable component of previous media used for the cultivation of these ciliates has been replaced by a mixture of trypticase, yeast nucleic acid and TEM-4T. Particle-bearing animals of stock 299 lambda, 138 mu, and 139 pi maintain their particles when cultivated in the medium, whereas particle-bearing animals of stock 51 kappa, 225 kappa and 114 signia do not. With the exception of stock 92 (syngen 3) the medium appears to be selective in its ability to support the growth of animals of the even- but not odd-numbered syngens of P. aurelia. Maintenance of the particles was dependent only to a small degree upon environmental conditions brought about by changes in pH and temperature. Division of the particles was found to be comparable with the division of the protozoan. Methods for the growth, maintenance and mass cultivation of particle-bearing P. aurelia are given in detail.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Calcium distribution in Amoeba proteus

A preliminary investigation of the distribution of cellular calcium in Amoeba proteus was undertaken. Total cellular calcium under control conditions was found to be 4.59 mmol/kg of cells. When the external Ca++ concentration is increased from the control level of 0.03 to 20 mM, a net Ca++ influx results with a new steady-state cellular calcium level being achieved in integral of 3 h. At steady state the amount of calcium per unit weight of cells is higher than the amount of calcium per unit weight of external solution when the external concentration of Ca++ is below 10 mM. At external Ca++ concentrations above this level, total cellular calcium approaches the medium level of Ca++. Steady-state calcium exchange in Amoeba proteus was determined with 45Ca. There is an immediate and rapid exchange of integral of 0.84 mmol/kg of cells or 18% of the total cellular calcium with the labelled Ca++. Following this initial exchange, there was very little if any further exchange observed. Most of this exchanged calcium could be eliminated from the cell with 1 mM La+++, suggesting that the exchanged calcium is associated with the surface of the cell. Increase in either the external Ca++ concentration of pH raise the amount of exchangeable calcium associated with the cell. Calcium may be associated with the cell surface as a co-ion in the diffuse double layer or bound to fixed negative sites on the surface of the cell. If Ca++-binding sites do exist on the cell surface, there may be more than one type and they may have different dissociation constants. The cytoplasmic Ca++ ion activity is probably maintained at very low levels.     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.