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PubMed2006

Relationship between testate amoeba (protist) communities and atmospheric heavy metals accumulated in Barbula indica (bryophyta) in Vietnam.

We studied the relationships between testate amoeba communities and heavy metal (Pb, Cd, Zn, Ni, Cu, Mn, and Fe) concentrations in the moss Barbula indica sampled at 29 sites in and around the city of Hanoi (Vietnam). Our first approach was to compare the heavy metal concentrations and testate amoeba variables between the city (zone 1) and the surrounding (zone 2). Mean moss concentrations of Pb, Cd, Zn, Ni, and Cu were significantly higher and testate amoeba species richness and abundance were significantly lower in zone 1 and the abundance of eight taxa differed significantly between the two zones. We then studied the correlation between heavy metals and testate amoebae. Species richness and abundance were correlated negatively to Pb concentration. Shannon H' was negatively correlated to both Pb and Cd. The abundance of several species was negatively correlated with Pb, Cd, Zn, and Ni; however, at the community level, Pb emerged as the only significant variable in a redundancy analysis. Our results suggest that testate amoebae are sensitive to and may be good bioindicators for heavy metal pollution, especially lead. Further research is needed to understand the causal relationships underlying the observed patterns.

Air Pollutants↗

PubMed2003

Effect of Rho family GTP-binding proteins on Amoeba proteus.

While there is a number of studies on the effects of Rho GTPases on the actin-based cytoskeleton in higher eukaryotes, studies in protozoans are rather limited. The problem seems to be intriguing since the structure of protozoan cytoskeletons is distinct from most vertebrate cells. By blocking endogenous Rho family proteins of highly motile Amoeba proteus with C3 transferase and antibodies against human RhoA and Rac1, we tried to assess the in vivo role of these proteins in amoebae. In migrating amoebae, both proteins are concentrated in the cortical layer and seem to colocalize with filamentous actin. Endogenous Rac1, but not RhoA, is accumulated in the perinuclear cytoskeleton. Blocking Rac- or Rho-like proteins caused distinct and irreversible changes in the locomotive shape of the examined amoebae and significant inhibition of their migration. Amoebae microinjected with anti-Rac1 antibodies were contracted, shortened, and developed only few wide pseudopodia. More pronounced changes were observed in cells treated with anti-RhoA antibodies. They exhibited an atypical locomotion not leading to their effective displacement. After treatment with 50 microg of C3 transferase per ml, cells rapidly contracted and almost completely rounded up, became refractile with the granules beaten into a dense mass, detached from the surface and died. Ten times lower concentration of the enzyme caused similar changes as the inhibition of endogenous RhoA-like protein. These results indicate that Rho family-based regulation plays a key role in amoebic migration.

Amoeba↗

PubMed2005

Characterisation of the Rac/PAK pathway in Amoeba proteus.

Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5-6) than the control cells (9-10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells.

Actins↗

PubMed1992

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.

Actins↗

PubMed2006

Ecology of testate amoebae from mires in the Central Rhodope Mountains, Greece and development of a transfer function for palaeohydrological reconstruction.

Testate amoebae are useful environmental indicators in ecological and palaeoecological studies from peatlands. Previous quantitative studies have focused on the peatlands of Northern and Central Europe, North America, and New Zealand and have considered a relatively restricted variety of peatland types, mostly ombrotrophic or Sphagnum-dominated while more minerotrophic fens have been less studied. Here we present the first quantitative ecological study of testate amoebae from four small mesotrophic fens (pH 5.5-8.1) in the Elatia Forest, northern Macedonia province, Greece. Relationships with the environmental data were investigated using redundancy analysis and mantel tests. Transfer function models were derived using a variety of techniques. Results demonstrate that as for Sphagnum-dominated mires hydrology is the most important control on amoebae community composition. Transfer function models should enable water tables to be predicted within 2.5 cm, when data selection is used this is reduced to less than 2 cm. pH is also an important environmental control on testate amoebae communities, a transfer function model enables pH prediction within 0.4 pH units. The hydrological transfer function is the best performing such model yet produced in terms of prediction error. This study provides new data on the ecology of testate amoebae in fens, and the transfer function models should allow quantitative palaeohydrological reconstruction.

Amoeba↗

PubMed2001

Bacterial infections of free-living amoebae.

Free-living amoebae are a diverse group of ubiquitous unicellular organisms, some of which cause severe central nervous system infections and keratitis. However, the focus of research has shifted from the direct pathogenic effects of free-living amoebae towards their role as carriers of pathogenic bacteria. Large outbreaks of legionellosis with numerous fatal cases, both in hospitals and in the community, appear to be the visible tip of the iceberg of complex relationships between amoebae and bacteria in biofilms. The recognition of amoebae as reservoirs and vehicles for bacterial spread leads us to public health issues such as the development of pathogenicity, antibiotic resistance, quality of public water supplies, housing standards, sanitation and decontamination measures. In this review we discuss bacterial infections of free-living amoebae from both a "biological" and general "infection control" point of view.

Acanthamoeba↗

PubMed1964

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS. 3. FURTHER STUDIES ON THE NATURE OF THE DNA--CONTAINING ELEMENTS.

The application of electron microscope autoradiography to Amoeba proteus cells labeled with tritiated thymidine has permitted the identification of morphologically distinct particles in the cytoplasm as the sites of incorporated DNA precursor. The particles correspond to those previously described from light microscope studies, with respect to both H(3)Tdr incorporation and distribution in centrifugally stratified amoebae. Ingested bacteria differ from the particles, in morphology as well as in the absence of associated label. Attempts to introduce a normal particle labeling pattern by incubating amoebae with labeled sediment derived from used amoeba medium failed. The resultant conclusion, that the particles are maintained in the amoeba by self-duplication, is supported by the presence of particles in configurations suggestive of division.

Amoeba↗

PubMed1959

Nuclear synthesis of cytoplasmic ribonucleic acid in Amoeba proteus.

The enucleation technique has been applied to Amoeba proteus by several laboratories in attempts to determine whether the cytoplasm is capable of nucleus-independent ribonucleic acid synthesis. This cell is very convenient for micrurgy, but its use requires a thorough starvation period to eliminate the possibility of metabolic influence by food vacuoles and frequent washings and medium renewal to maintain asepsis. In the experiments described here, amoebae were starved for periods of 24 to 96 hours, cut into nucleated and enucleated halves, and exposed to either C-14 uracil, C-14 adenine, C-14 orotic acid, or a mixture of all three. When the starvation period was short (less than 72 hours), organisms (especially yeast cells) contained within amoeba food vacuoles frequently showed RNA synthesis in both nucleated and enucleated amoebae. When the preperiod of starvation was longer than 72 hours, food vacuole influence was apparently negligible, and a more meaningful comparison between enucleated and nucleated amoebae was possible. Nucleated cells incorporated all three precursors into RNA; enucleated cells were incapable of such incorporation. The experiments indicate a complete dependence on the nucleus for RNA synthesis. The conflict with the experimental results of others on this problem could possibly stem from differences in culture conditions, starvation treatment, or experimental conditions. For an unequivocal answer in experiments of this design, ideally the cells should be capable of growth on an entirely synthetic medium under aseptic conditions. The use of a synthetic medium (experiments with A. proteus are done under starvation conditions) would permit, moreover, a more realistic comparison of metabolic capacities of nucleated and enucleated cells.

Adenine↗

PubMed1975

Cytoplasmic filaments and cellular wound healing in Amoeba proteus.

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 mum, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.

Amoeba↗

PubMed1999

Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis.

Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lack immunity to B. mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoeba by immunofluorescence. Sera from adults contained titers of 1/64-1/256 of anti-B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar to those in adults were seen in serum of 1- to 5-year-old children.

Acanthamoeba↗

PubMed1982

Cocultivation of Legionella pneumophila and free-living amoebae.

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.

Amoeba↗

PubMed1989

Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake.

Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.

Air↗

PubMed1992

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.

Amoeba↗

PubMed1998

Comparison of free-living amoebae in hot water systems of hospitals with isolates from moist sanitary areas by identifying genera and determining temperature tolerance.

Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44 degrees C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53 degrees C.

Acanthamoeba↗

PubMed1997

Interaction of Mycobacterium avium with environmental amoebae enhances virulence.

Environmental mycobacteria are a common cause of human infections. Recently, contaminated domestic water supplies have been suggested as a potential environmental source of several mycobacterial diseases. Since many of these mycobacterial species replicate best intracellularly, environmental hosts have been sought. In the present study, we examined the interaction of Mycobacterium avium with a potential protozoan host, the water-borne amoeba Acanthamoeba castellanii. We found that M. avium enters and replicates in A. castellanii. In addition, similar to that shown for mycobacteria within macrophages, M. avium inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae. In order to determine whether growth of M. avium in amoebae plays a role in human infections, we tested the effects of this growth condition on virulence. We found that growth of M. avium in amoebae enhances both entry and intracellular replication compared to growth of bacteria in broth. Furthermore, amoeba-grown M. avium was also more virulent in the beige mouse model of infection. These data suggest a role for protozoa present in water environments as hosts for pathogenic mycobacteria, particularly M. avium.

Amoeba↗

PubMed1969

Contamination of virological tissue cultures with a species of free-living soil amoeba.

Overwhelming contamination of a continuous cell line with a free-living soil amoeba of the genus Hartmannella is reported. The significance of the finding is discussed because of recent interest shown in the role of this and related species of amoebae in disease of humans. The methods used to detect and identify the amoeba are described and some examples of the amoebae are shown by photomicrographs. The use of metronidazole to eliminate the amoeba from tissue culture is also described. This procedure has proved successful in practice although the empirical basis for it has not been confirmed by direct assay. Consequently, there is a need to test possible alternative chemical regimens.

Air Microbiology↗

PubMed1981

The appearance of carbohydrate-rich material in the developing Golgi apparatus of amoebae.

The silver proteinate reaction was used to stain carbohydrate-rich substances in normal Amoeba proteus and in the developing Golgi apparatus of renucleated amoebae. Normal cells contained stained material, which probably is glycoprotein, in the cell surface, cisternae at the concave pole of the Golgi apparatus, and cytoplasmic vesicles and vacuoles. Previous radioautographic studies had shown tht glycosylation occurs in the Golgi apparatus, and that material in the Golgi apparatus is precursor to the cell surface. Amoebae were enucleated for 5 d, which results in a decline of the Golgi apparatus, the disappearance of the glycoprotein-containing cisternae preceding that of the rest of the organelle. A new nucleus was then transplanted into the enucleate amoebae, bringing about the regeneration of the Golgi apparatus. small curved cisternae that appeared 30 min after renucleation lacked staining with silver proteinate. By 1 h after renucleation, however, the content of cisternae toward the concave poles of Golgi bodies stained with silver proteinate. The Golgi apparatus in cells fixed 6 h and 1 d after operation resembled that of normal amoebae in both morphology and staining pattern. The results suggest that the developing Golgi apparatus acquired the capacity to participate in assembly of cell-surface material within 1 h after renucleation. This occurred before development of the normal enzymic activity of the Golgi apparatus was completed.

Amoeba↗

PubMed1984

A cell-cycle-phase-specific mutant of amoeba.

The treatment of Amoeba indica with ethylmethanesulphonate (EMS) at early S, late S and late G2 phases of the cell cycle leads to the production of mini amoeba cells in the G2 period. Among them, only a few of the mini cells that originated from EMS treatment at early S phase have been found to be viable and to give rise to stable clones. These mini amoebae show stable and altered characteristic features in cell size, structure, membrane properties, cell-cycle timing and the patterns of macromolecular syntheses as compared to the parental cells. It is suggested that the mini amoeba cell is a size mutant that has a cell-cycle-phase-specific origin. The finding is discussed in relation to preferential mutagenic action involving the functional state of DNA leading to the production of viable mutant amoebae.

Amoeba↗