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Effect of bacteria and amoebae on rhizosphere phosphatase activity.

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.

Journal Article↗

Immunity to pathogenic free-living amoebae: role of humoral antibody.

Pathogenic free-living amoebae are common in nature, but few clinical infections by these amoebae have been reported. This has prompted studies of host susceptibility factors in humans. A survey of normal human sera from three New Zealand Health Districts was made; antibodies to pathogenic free-living amoebae were found in all sera, with titers ranging from 1:5 to 1:20 for Naegleria spp. and from 1:20 to 1:80 for Acanthamoeba spp. The antibodies belonged mainly to immunoglobulin G and immunoglobulin M classes. The presence of a specific neutralizing factor against Acanthamoeba spp. but not Naegleria spp. was demonstrated. Possible protective mechanisms are discussed.

Amebiasis↗

Immunity to pathogenic free-living amoebae: role of cell-mediated immunity.

The role of cell-mediated immunity in defense against pathogenic free-living amoebae was examined. Both the in vitro macrophage inhibition test and the in vivo delayed hypersensitivity test showed responses to both heterologous and homologous antigens, although homologous systems were the most efficient. It is suggested that exposure to nonpathogenic species of free-living amoebae can stimulate the immune system to be effective against pathogenic species. The significance of cell-mediated immunity as a defense against invasion by pathogenic free-living amoebae is discussed.

Amebiasis↗

Resistance of highly pathogenic Naegleria fowleri amoebae to complement-mediated lysis.

Weakly pathogenic and nonpathogenic Naegleria spp. are readily lysed by human and guinea pig complement. Highly pathogenic Naegleria fowleri are resistant to complement-mediated lysis. Electrophoretic analysis of normal human serum (NHS) incubated with pathogenic or nonpathogenic Naegleria spp. demonstrates that amoebae activate the complement cascade, resulting in the production of C3 and C5 complement cleavage products. To determine whether surface constituents play a role in resistance to complement lysis, trophozoites of Naegleria spp. were subjected to enzymatic treatments prior to incubation in NHS. Treatment of trophozoites with papain or trypsin for 1 h, but not with neuraminidase, increased susceptibility of highly pathogenic Naegleria fowleri to complement lysis. Treatment of trophozoites with actinomycin D or cycloheximide during incubation with NHS or pretreatment with various protease inhibitors for 4 h did not increase the susceptibility of N. fowleri amoebae to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appears to account for the increased resistance of N. fowleri amoebae to complement-mediated lysis.

Animals↗

Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease.

We report the identification of a set of Legionella pneumophila genes that encode products with homology to proteins of the type II general secretion pathway of gram-negative bacteria. A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease. This strain was unable to multiply within the free-living amoeba Acanthamoeba castellanii yet was able to kill HL-60-derived macrophages. Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway.

Acanthamoeba↗

Growth and bacteriolytic activity of a soil amoeba, Hartmannella glebae.

A soil amoeba, Hartmannella glebae, could grow on a variety of gram-positive and gram-negative bacteria, although the rate of growth was faster in the presence of gram-negative bacteria. The amoeba, however, could not use yeasts, molds, or a green alga as a nutritional source. The extract prepared from amoebae grown in the presence of Aerobacter aerogenes and Alcaligenes faecalis could lyse intact cells and cell walls of many gram-positive bacteria at different rates. The spectrum of lytic activity was similar to that of egg-white lysozyme, with the exception that several species and strains of Bacillus, Micrococcus, and Staphylococcus were resistant to lysozyme and susceptible to the extract. The gram-negative bacteria tested were resistant.

Bacteria↗

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

DNA↗

Digital life behavior in the Amoeba world.

Amoeba is a computer model designed to facilitate the study of the origin and evolution of digital life. Specifically, an initially disordered system, consisting of random sequences of machine instructions, self-organizes into an ordered system containing self-replicating programs. The current version of Amoeba broadens the original system's capability by using a basis set of 32 machine instructions that is computationally universal. In addition, Amoeba uses a set of 64 address labels, each of which is randomly assigned to a machine instruction each time a sequence is randomly created. This eliminates the constraint that occurs when the complements of predefined codons are used for addressing. A more open-ended system results because programs can now form subroutines that are arranged in an arbitrary manner.

Animals↗

Cyclic AMP waves during aggregation of Dictyostelium amoebae.

During the aggregation phase of their life cycle, Dictyostelium discoideum amoebae communicate with each other by traveling waves of cyclic AMP. These waves are generated by an interplay between random diffusion of cyclic AMP in the extracellular milieu and the signal-reception/signal/relaying capabilities of individual amoebae. Kinetic properties of the enzymes, transport proteins and cell-surface receptor proteins involved in the cyclic AMP signaling system have been painstakingly worked out over the past fifteen years in many laboratories. Recently Martiel & Goldbeter (1987) incorporated this biochemical information into a unified mathematical model of communication among Dictyostelium amoebae. Numerical simulations of the mathematical model, carried out by Tyson et al. (1989), agree in quantitative detail with experimental observations of cyclic AMP traveling waves in Dictyostelium cultures. Such mathematical modeling and numerical experimentation provide a necessary link between detailed studies of the molecular control mechanism and experimental observations of the intact developmental system.

Cyclic AMP↗

Naegleria fowleri: a free living amoeba of emerging medical importance.

Naegleria fowleri, a free-living amoeba is ubiquitous and word-wide in distribution. Infection is due to inhalation or aspiration of aerosols containing cysts found in the environment. Of late, the amoeba is emerging as a pathogen of medical importance causing primary amoebic meningoencephalitis (PAM) in humans. The diagnosis of the condition is mainly parasitic which depends on the detection and identification of Naegleria trophozoites in the cerebro-spinal fluid (CSF) or biopsied brain tissue. Serological tests are not useful in the diagnosis of PAM. Most cases are fatal and various amoebicidal agents have been tried unsuccessfully. The present paper provides a review of the recent information on the biology and epidemiology of the disease caused by the amoeba Approaches in the diagnosis, pathophysiology and treatment of the condition are also discussed.

Amebiasis↗

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae.

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.

Acanthamoeba castellanii↗

Entamoeba histolytica-like amoebae occurring in man.

Six Entamoeba histolytica-like amoebae of the Laredo-type are compared with classical E. histolytica strains with regard to morphology, temperature-tolerance, response to hypotonic solutions, antigenic make-up, pathogenicity, sensitivity to drugs, and biochemistry. Sharp discontinuities are shown to exist between the 2 groups in some parameters, sufficient, under certain conditions, to justify creation of a new species designation for the Laredo-type amoebae. Nevertheless, this action is not recommended at the present time because of the small number of "aberrant" strains so far isolated, and because of the limited comparative studies so far carried out.Entamoeba moshkovskii is compared with the Laredo-type E. histolytica. No consistent distinction can be made between the 2 groups except for the sources of the strains-the former is free-living and the latter parasitic. It is therefore suggested that E. moshkovskii may actually be a parasite of the Laredo type, only incidentally encountered in free-living environments because of its wide temperature and tonicity tolerances. Since, here too, only limited studies are so far available, it is suggested that the term "moshkovskii-like E. histolytica" be used until sufficient data are accumulated to assign these amoebae to a more precise location in the taxonomic scheme.

Amebicides↗

Cyclic nucleotide-dependent phosphorylation in Dictyostelium discoideum amoebae.

Stimulation of Dictyostelium discoideum amoebae with cAMP was found to induce the specific phosphorylation of a 47,000 molecular weight protein (pP47). This cellular response to cAMP was developmentally regulated. It was first detected in 3 1/2-h starved cells and appeared to persist throughout the aggregation phase of the cells' life cycle. pP47 phosphorylation was specifically induced by cAMP in that amoebae did not respond to stimulation with 5'-AMP, folic acid, Ca2+, and/or the Ca2+ ionophore A23187. cGMP could elicit pP47 phosphorylation but only at high concentrations. Phosphorylation of pP47 in response to cAMP occurred rapidly (within 5 s). The length of time for which it remained phosphorylated depended upon the concentration of the stimulus. With 10(-6) M cAMP, pP47 was phosphorylated for less than 4 min. If amoebae were stimulated with 10(-4) M cAMP, over 30 min were necessary before pP47 was dephosphorylated. Once dephosphorylated, pP47 could again be phosphorylated upon reapplication of the cAMP stimulus.

Calcimycin↗

Dictyostelium amoebae can differentiate into spores without cell-to-cell contact.

Amoebae of sporogenous mutants of Dictyostelium discoideum can differentiate into stalk cells and spores in the absence of normal morphogenesis when spread on agar containing cyclic-AMP. The efficiency of differentiation is improved when the amoebae are incubated as submerged monolayers in plastic petri dishes. Under these conditions spore formation is density dependent and hence requires some form of cellular interaction. To determine whether this interaction involves direct cell-cell contact we have made time-lapse films of cells differentiating at intermediate density. These films show that amoebae can develop into spores without making contact with any other cells. In addition, although some cells do divide during incubation, division is not necessary for spore formation. At higher densities small aggregates form which give rise to mixtures of stalk cells and spores. There is no detectable patterning of the two cell types within such aggregates.

Dictyostelium↗

Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae.

The mechanisms by which bisphosphonate drugs inhibit osteoclast-mediated bone resorption are unclear. Effects of bisphosphonates on cellular enzymes, metabolic pathways, and osteoclast morphology have previously been described and could culminate in a generalized cytotoxic effect or a decreased capacity of osteoclasts to resorb bone. Recent studies of the structure-activity relationship for the bisphosphonate side chain indicate, however, that at least the newer generations of nitrogen-containing bisphosphonates probably act by binding to a specific target at a site that is complementary in structure to the bisphosphonate side chain. We have previously proposed that such a target for bisphosphonates is also present in amoebae of the cellular slime mold Dictyostelium discoideum, because growth of this microorganism is inhibited by a wide range of bisphosphonates in a manner that closely reflects the antiresorptive potencies of the bisphosphonates in vivo. We have added support for this view by examining the potency towards Dictyostelium of bisphosphonates in which slight changes in the structure of the side chain or conformational restrictions to the side chain have marked effects on antiresorptive potency. The changes in the side chain that affected the in vivo antiresorptive potency of the bisphosphonates consistently affected in a similar manner the potency of the bisphosphonates as inhibitors of the growth of Dictyostelium amoebae. These observations confirm that bisphosphonate drugs have a molecular target that is common to both Dictyostelium amoebae and osteoclasts.

Animals↗

Vexillifera expectata sp. n. and other non-encysting amoebae isolated from organs of freshwater fish.

Four strains of non-encysting amoebae were isolated from organs of freshwater fishes and characterized using light and electron microscope. Morphology of three clonal strains was consistent with amoebae which had already been described from water habitats. Two strains, one isolated from kidney tissue of common goldfish, Carassius auratus (Linnaeus, 1758), and the second one from brain of chub, Leuciscus cephalus Linnaeus, 1758, were identified with Vannella platypodia (Gläser, 1912) Page, 1976. Both strains were identical, except for the length of glycostyles. The strain isolated from the liver of perch, Perca fluviatilis (Linnaeus, 1758), was assigned to the genus Vexillifera Schaeffer, 1926 as Vexillifera expectata sp. n. The taxonomic position of the fourth non-encysting strain could not be safely established, although it shares some trophic cell structures with protostelids (Protostelia, Eumycetozoea). We present its detailed description here also to demonstrate that amoeba stages of this type of organisms are capable to infect fishes.

Amoebida↗

Cytoplasmic DNA synthesis in Amoeba proteus. I. On the particulate nature of the DNA-containing elements.

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 micro particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H(3)-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.

Amoeba↗

Cytoplasmic DNA synthesis in Amoeba proteus. II. On the behavior and possible nature of the DNA-containing elements.

Nucleic acid-containing particles in the cytoplasm of Amoeba proteus (cf. reference 1) were counted after acridine orange staining. The number of particles per ameba was found to be correlated with cell age and size. Fresh daughters had a mean particle number of 5400, whereas predivision amebae contained around 11,000 particles. Amebae from two other strains contained similar particles. The particles were found to be clustered in fasted cells and redispersed after feeding. A marked increase in the particle population was noted in anucleate fragments. These results, together with those previously presented, suggest that the particles multiply intracellularly. Their nature and their relationship to previous work on nucleic acid labeling in Amoeba are discussed.

Amoeba↗