Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Studies of Blastocystis hominis

Blastocystis hominis, grown in Boeck-Drbohlav culture medium, modified by the omission of rice starch and the addition of 20% human serum and mineral oil cover to the Locke's solution overlay, can assume 3 morphologic forms. In the absence of human serum the vacuolated form, which divides by binary fission predominates. In medium with high serum content the granular form appears, with 3 types of granules. Spheroid or more elongate cytoplasmic granules predominate. In older organisms, lipid granules are found either in the peripheral cytoplasm or in the central vacuolar space. In occasional cells, variable numbers of reproductive granules develop in the central vacuolar space. These latter granules are released from the organism and give rise to typical B. hominis cells. The 3rd form, the ameba form, appears in small numbers in older cultures and in those treated with antibiotics. Ameba forms feed on bacteria and have slow pseudopodial activity. Exposure to oxygen causes rapid damage to cell membrane, with resulting leakage and collapse.     

The Cell Cycle and Induced Amitosis in Acanthamoeba*

The small soil ameba, Acanthamoeba castellanii strain HR, with a mean generation time of 18 hr in axenic culture, requires 24 min for nuclear mitosis. This is followed immediately by nuclear DNA synthesis that is initiated in late telophase and lasts for 20 min. Evidence is presented that amitosis can be induced in susceptible amebas only during a portion of the cell cycle.     

Entamoeba Clone‐Recognition Experiments: Morphometrics,Aggregative Behavior,and Cell‐Signaling Characterization

Studies on clone‐ and kin‐discrimination in protists have proliferated during the past decade. We report clone‐recognition experiments in seven Entamoeba lineages (E. invadens IP‐1, E. invadens VK‐1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM‐1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell‐surface area) and documented how they differed statistically from one another (as per single‐variable or canonical‐discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix‐cell‐line cultures between closely‐related (E. invadens IP‐1 vs. E. invadens VK‐1:NS) or distant‐phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same‐clone members. Third, we identified six putative cell‐signals secreted by the amebas (RasGap/Ankyrin, coronin‐WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress‐induced encystation. To our knowledge, this is the first multi‐clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell‐signaling secretion in the context of clone‐recognition. Protists allow us to study cell–cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity.     

Fine Structure of a Marine Ameba Associated with a Blue-Green Alga in the Sargasso Sea*,†

SYNOPSIS An ameba, bearing a fringe of scales on the plasmalemma surface, dwells among the filaments of the colonial, blue-green alga Trichodesmium thiebautii (Sournia), and preys upon bacteria growing within the colony. The cytoplasm is clearly differentiated into a fine fibrillar ectoplasm at the periphery of the cell and a central endoplasm containing most of the membranous organelles. The nucleus contains a spheroidal nucleolus which is centrally located, and a double membrane containing pores. The tubular mitochondria, microbodies, lysosomes, and endoplasmic reticulum are typical for protozoa. The Golgi apparatus consists of an array of elongate flattened cisternae. One surface is associated with a fine fibrillar layer and the opposite surface contains electron-dense vesicles (perhaps primary lysosomes) and scale-containing vesicles that appear to be the origin of the scales deposited on the plasma membrane. Three kinds of bacteria-containing vacuoles are presnt: (a) vacuoles surrounded by 3 membranes and containing bacteria that are either healthy or in an early stage of digestion, (b) singlemembrane vacuoles which are food vacuoles that become converted to digestive vacuoles, and (c) larger vacuoles resembling those in (b) which contain prey in an advanced stage of digestion. The presence of amebae within pelagic algal communities provides further evidence for the diversity of their habitats in the ocean.     

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

NACM, A Cytopathogenic Protein from Naegleria gruberi, EGs; Purification, Production of Monoclonal Antibody, and the Immunoidentification of a Product that Develops in NACM-treated Vertebrate Cell Cultures

The story of NACM involves the discovery of a deleterious response of cultured vertebrate cells to a component in cell-free lysates prepared from free-living amebae of the genus Naegleria; hence the acronym NACM derived from Naegleria ameba cytopathogenic material. The cellular reaction is the basis for the biological assay that has been fundamental in the study of the action of NACM in a variety of cell cultures. It also has been used in the determination of the physical characteristics, and to monitor the behavior of NACM during isolation procedures. All findings are compatable with the conclusion that NACM is a 35 Kd protein. Recently, the use of monoclonal antibodies (MAbs) prepared to amebae-derived purified NACM have resulted in visual display of a product that develops exclusively in NACM-treated cells. That cellular product is shown to be related to NACM by its immunostaining reaction with the MAb; the relationship of the MAb with NACM is demonstrated by its ability to neutralize the biological activity of NACM, and as an immunostain, to react with purified fractions of NACM and with whole amebae. The combination of these observations describes a unique set of interactions in which NACM, an amebic component, identified as a protein, has characteristics of an infectious agent when introduced into cultures of avian and mammalian cells.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.     

The ameba Balamuthia mandrillaris feeds by entering into mammalian cells in culture

Microscopic observations of live cultures of the pathogenic ameba Balamuthia mandrillaris and mammalian cells showed that amebic feeding involved the invasion of the pseudopodia, and/or the whole ameba into the cells. The ameba, recognized by their size and flow of organelles in the cytosol, was seen to extend the tip of a pseudopodium into the cytoplasm of a cell where it moved about leaving visible damage when retracted. In rounded cells, whole amebas were seen to enter into and move around before exiting a cell and then remain quiescent for hours. The invaded mammalian cells retained their turgidity and excluded vital dyes until only their denuded nuclei remained. The cytoplasm of the cells was consumed first, then the nuclei, but not their mitotic chromosomes. The feeding pattern of four isolates of B. mandrillaris, two from humans and two from soil samples, was by amebic invasion into the mammalian cells. The resulting ameba population included cysts, amebas on the surface, and free-floating amebas as individuals or in dense-packed clusters. There was no morphologic indication of a cytopathic change in the mammalian cells before their invasion by the amebas. Feeding by cell invasion is a distinctive feature of B. mandrillaris.