Intermediate Metabolism of Carbohydrates in Mayorella palestinensis

SYNOPSIS. The mechanism of carbohydrate metabolism was studied in homogenates of the ameba, Mayorella palestinensis. The utilization of intermediates with the formation of lactic acid as the end product of the anaerobic breakdown of the carbohydrates was established. Evidence was obtained for the presence of the main enzymes associated with the Embden-Meyerhof glycolytic scheme. The activity of hexokinase and phosphorylase was ascertained in a M. pakstinensis homogenate. These enzymes have not yet been found among other soil amebas grown in axenic cultures.     

Observations on acid phosphatase in Mayorella palestinensis

Acid phosphatase activity has been studied in the ameba Mayorella palestinensis. Optimum activity of the enzyme was found to be at a pH of 3.2. The enzyme is inhibited by fluoride ion, but is not sensitive to Mg⁺⁺. The activity was found to be correlated with age of culture. Two maxima have been obtained, one from cultures in the logarithmic phase, and the other during the period of maximal cell encystation. These results suggest that acid phosphatase play an important role in cell metabolism during growth and differentiation processes of this ameba.     

Effects of Chemical Substances on Rate of Locomotion in the Amoeba Mayorella penardi1

The relationship between the locomotive velocity of amoeba which had not been fed for 24 h and the concentration of the test solutions was examined. With solutions of L-amino acids, protein substances, and alcian blue 8GS, an increase in velocity began at very low concentrations, reaching a maximum at a higher concentration and as the concentration increased further, the velocity fell to zero. In contrast, no increase was observed with D-glutamic acid and β-alanine. Moreover, the velocity of well fed amoebae did not increase significantly even in L-amino acid solution. These results may suggest a correlation between orthokinesis and amoeboid phagocytosis.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

A Lecithinase from the Amoeba Hartmannella rhysodes*

SYNOPSIS A lipogenic toxin produced by the amoeba Hartmannella rhysodes, Fernald strain, made mammalian cells in culture round up and fill with fat droplets. From this toxin an enzyme was obtained with lecithinase and lysolecithinase activities. This enzyme is different from any isolated elsewhere. When purified enzyme was added to strain L mouse fibroblasts in culture, the cells rounded up and became fatty. Much of the lipogenic activity of the original toxin can be ascribed to this enzyme.     

Carbohydrate Utilization by the Soil Amoeba Hartmannella castellanii

SYNOPSIS. Carbohydrate utilization by 9 strains of Hartmannella castellanii has been studied by growing the amoebae in a chemically defined medium which did not support growth without an added energy source. Strains differed in the utilization of sucrose, raffinose, melibiose and mannitol. The strains which did not use sucrose for growth were shown to metabolize this sugar: ¹⁴CO₂ was produced and ¹⁴C incorporated into TCA isoluble compounds when the amoebae were grown in the presence of radioactive sucrose.     

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.     

Glucosidase Activity in Acanthamoeba (Mayorella) palestinensis. The Effect of Glucose and Natural Glucosides on α- and β-Glucosidases

SYNOPSIS. Acanthamoeba ( Mayorella ) palestinensis produces high basal levels of α- and β -glucosidases, the latter being much more active than the former. Glucose, an essential growth substance, has a dual effect on the glucosidase activity. Growth concentrations (1%) of glucose inhibit, while low levels elevate the activity of both enzymes. Natural α-glucosides support growth in the same manner as glucose and raise the activity of both enzymes to the same extent. β -glucosides, on the other hand, are weak growth substrates, but stronger inducers, especially for β -glucosidase activity. The role of the glucosidases in the over-all metabolism of the ameba is discussed.     

The Feeding of Amoeba proteus on Paramecium aurelia*

SYNOPSIS. Density of prey (Paramecium aurelia) and predator (Amoeba proteus) were varied while volume of inorganic medium was kept constant. Variations in density of prey had little effect on the rates of feeding and reproduction of the amoebae; but with increasing predator density the amoebae captured the paramecia less rapidly and ingested fewer before dividing, altho division size did not change appreciably. Therefore, amoebae of a low density population with a constant food supply carry more nutritional reserves from generation to generation than do those in a denser population.     

The Contractile Vacuole of Amoeba proteus. II. Numerical Analysis of the Steady-State Hypothesis*

SYNOPSIS. At temperatures below 15 C, the contractile vacuole cycle of Amoeba proteus includes a presystolic plateau. The hypothesis attributing this plateau to a steady-state equilibrium between active filling processes and osmotic losses of water from the vacuole into the cytoplasm has been expressed in an equation predicting vacuolar diameter as a function of time for the later part of the cycle. Computer-generated model cycles have been compared with actual recorded cycles at 15 C, 10 C and 5 C and conditions of best fit were determined. Statistical analysis shows that recorded cycles are quite compatible with the steady-state hypothesis.     

Activation of adenylate cyclase in Acanthamoeba palestinensis

Preincubation of Acanthamoeba palestinensis homogenates in 0.25M sucrose-TM (2mM MgSO4 and 5mM Tris-HCl, pH 7.4) at 0 degree C for increasing periods of time up to 3 h, leads to a progressive increase in the activity of adenylated cyclase. In contrast, preincubation of isolated membrane fractions enriched in enzyme activity in the same medium results in no activation. However, preincubation of membrane fractions in medium containing a high density of sugars (sucrose, glucose or fructose) mimics the activation obtained with homogenates. The high density sugar activation is time and temperature dependent, and reversible upon return to a low density medium. The high osmotic pressure of the sugars utilized may be a factor, since high concentrations of the sucrose polymer, Ficoll, which has low osmotic activity, causes not activation. Soluble activators, protein synthesis and changes in cyclic nucleotide phosphodiesterase activity were all eliminated as possible effectors of the apparent activation of adenylate cyclase. In contrast to mammalian adenylate cyclase, the endoplasmic reticulum localized enzyme of Acanthamoeba is inhibited by NaF and is unaffected by GTP, adenosine, epinephrine, prostaglandin E1, propranolol, and meclofenamic acid. These data indicate that the adenylate cyclase of Acanthamoeba is structurally different from that of most mammalian cells.     

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.     

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.     

Photosensitized inactivation of Acanthamoeba palestinensis in the cystic stage

AIMS: To develop alternative approaches for medical and environmental control of pathogenic Acanthamoeba spp. by means of photodynamic treatment with a tetracationic Zn(II)-phthalocyanine (RLP068). METHODS AND RESULTS: Incubation of cyst cultures with RLP068 for 1 h caused an accumulation of readily detectable concentrations of the phthalocyanine, even at doses as low as 0.5 micromol l(-1). RLP068 exhibited no dark toxicity towards cysts up to 5 micromol l(-1) concentration. A decrease of c. 50% in cyst survival in comparison with controls was measured upon incubation of the cysts with 0.5 micromol l(-1) RLP068, followed by exposure to light (600-700 nm) for 20 min at a fluence rate of 50 mW cm(-2) (60 J cm(-2)). After incubation with 3 and 5 micromol l(-1) RLP068 and irradiation, the cysts lost their excystment ability as early as day 5 and up to day 10, and were clearly damaged when observed under an interference contrast microscope. CONCLUSIONS: These data indicate the promising use of RLP068 in phototreatment of diseases caused by pathogenic amoebae and in initial disinfection of wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and extensive photodamage may be induced in the highly resistant cystic stages by means of 600- to 700-nm light sources.     

Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.     

Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites

Aims:  To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments.
Methods and Results:  We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment.
Conclusions:  The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage.
Significance and Impact of the Study:  Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis , opening new perspectives for the disinfection of microbiologically polluted waters.