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.     

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.     

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.     

Specific Incorporation of Precursors into DNA by Feeding Labeled Bacteria to Paramecium aurelia

SYNOPSIS. Studies were carried out on the introduction of labeled precursors into the DNA of Paramecium aurelia (syngen 4, stock 51) by way of the bacteria that are used for food. A thymine-requiring strain of Escherichia coli (15 T⁻) was labeled by growth in either H³-methyl thymidine or 2-C¹⁴ bromouracil, washed free of the exogenous label, and fed to the paramecia. The tritium label from the bacteria was incorporated almost exclusively into the DNA of the paramecia, whereas it was much less specifically incorporated when introduced directly from the medium. The Cu label from bromouracil was also incorporated mainly into the DNA of the paramecia although a small amount appeared in RNA. The formation of labeled food vacuoles was followed. Food vacuoles were formed at a nearly constant rate, with the total number of vacuoles increasing throughout the cycle. The lifetime of the vacuoles was about 2.5 hours. Incorporation of the label into the DXA of the paramecia begins within a few minutes of the formation of the first labeled vacuole. DNA synthesis begins about 1.5 hr after the previous fission (total cell cycle about 5.8 hr) and progresses at a nearly constant rate throughout the remainder of the cycle.