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PubMed1977

Relationships between membranous organelles in amoebae studied by electron microscopic cytochemical staining.

The intracellular location of a variety of enzymes was studied in Amoeba proteus with the use of electron microscopic cytochemical methods, in an attempt to assess the relationships between different membranous organelles. One group of enzymes, including nucleoside diphosphatases (IDPase, UDPase, GDPase, ADPase), carbamoyl phosphatase, alkaline phosphatase, and BAXD oxidase was localized mainly in the rough endoplasmic reticulum, nuclear envelope, and convex side of the Golgi apparatus. Esterase activity had a similar localization except that the Golgi apparatus was "stained" throughout most of its extent. A second group of enzymes was found in Golgi cisternae and vesicles, and in come vacuoles. This group included acid phosphatase, thiamine pyrophosphatase, and aryl sulfatase. Some enzymes previously detected in cytoplasmic membranes of other cells, including glucose-6-phosphatase, showed little or no activity in amoebae. The results suggest that there are chemical similarities and probable functional relationships between the rough endoplasmic reticulum, the nuclear envelope, and the convex side of the Golgi apparatus. On the other hand, the concave pole of the Golgi apparatus, aggregates of smooth tubules and vesicles, and the cell surface appear more closely related to one another than to the endoplasmic reticulum and the convex side of the Golgi apparatus. The cytochemical similarity between the Golgi apparatus and certain vacuoles such as food vacuoles may reflect the role of the Golgi apparatus in the formation of lysosomes. The locations of reaction products of the various enzymes in amoebae are compared with observations reported for other cell types.

Alkaline Phosphatase↗
PubMed2006

Survey for the presence of specific free-living amoebae in cooling waters from Belgian power plants.

Free-living amoebae (FLA) are distributed ubiquitously in aquatic environments with increasing importance in hygienic, medical and ecological relationships to man. In this study, water samples from Belgian industrial cooling circuits were quantitatively surveyed for the presence of FLA. Isolated, thermotolerant amoebae were identified morphologically as well as using the following molecular methods: enzyme-linked immunosorbent assay and isoenzyme electrophoresis and PCR. Thermophilic amoebae were present at nearly all collection sites, and the different detection methods gave similar results. Naegleria fowleri was the most frequently encountered thermotolerant species, and concentrations of thermotolerant FLA were correlated with higher temperatures.

Amoeba↗
PubMed1999

Isolation and identification by partial sequencing of the 18S ribosomal gene of free-living amoebae from necrotic tissue of Basilliscus plumifrons (Sauria: Iguanidae).

A 3-year-old Basiliscus plumifrons developed a necrotic lesion on the tail resulting from nodules of unknown etiology. Investigation of necrotic tissue revealed several gram-negative bacteria as well as three different species of free-living amoebae. The amoebae were identified by morphological characters as belonging to the genera Acanthamoeba, Echinamoeba, and Naegleria, respectively. Partial sequencing of the 18S ribosomal gene was performed for reliable systematic determination. Two of the isolates showed thermotolerance. No isolate was growable in conventional liquid media, but the Acanthamoeba strain readily grew on a human cell line (HEp2). It remains unclear whether the amoebae fed on the coexisting bacteria or on host tissue.

Acanthamoeba↗
PubMed2003

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus.

Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.

Actins↗
PubMed2004

Polyamines biosynthesis and oxidation in free-living amoebae.

In this paper we describe the polyamine biosynthesis and oxidation processes, giving an overview about recent results in free-living Amoebae. The protozoa polyamine levels are different in comparison with mammalian cells. Also, the polyamine levels in protozoa cells change if these species are pathological or not for the human beings. All the amoeba strains show high concentrations of 1,3-diaminopropane (DAP), spermidine and acetylspermidine while spermine is absent. In these amoeba a considerable polyamine oxidase activity has been found, which acts on N8-acetylspermidine, but not on free polyamines. This enzyme is responsible, together with polyamine acetylase, of DAP synthesis whose function is not well known.

Amoeba↗
PubMed1986

Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae.

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus Calmette-Guérin caused direct cytolysis of the pathogenic free-living amoeba, Naegleria fowleri, in vitro. Amoebicidal activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibody-dependent cellular cytotoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable.

Amoeba↗
PubMed1976

A study of the change in DNA synthesis of S phase cells treated with N-methyl-N-nitrosourethane: a study using Amoeba proteus as a single cell model.

The present experiments using Amoeba proteus as a single cell model show that DNA synthesis continues during and after exposure of S phase cell to N-methyl-N'-nitrosourethane (MNU). At sublethal dose levels which caused long division delays, division and growth abnormalities and mutations, the amount of [3h] thymidine ([3h]Tdr) incorporated was decreased by 20-30%; at dose levels which killed all S phase cells it was inhibited by up to 90%. There was a direct correlation between the dose of MNU used and the degree of inhibition of [3H]Tdr incorporated. The effect was rapid, mainly taking place within 20 min of treatment. Amoeba heterokaryons (HKs) were used to examine the rate of DNA synthesis of treated and untreated nuclei in the same cytoplasm, i.e. where the nuclei would have the same [h]tdr intake, the same thymidine kinase (TK) activity and the same endogenous precursor pools. Direct comparison of the nuclear DNA synthetic activity in this way revealed less difference between treated and untreated nuclei than comparisons made using the nuclear grain counts from treated and untreated amoebae. This suggested that much of the decrease in [3H]Tdr incorporation by MNU-treated S phase cells was due to a change in the cytoplasm and/or the cell membrane, rather than to nuclear damage. Thus MNU-treated nuclei were able to synthesize DNA at a near normal rate when they could draw on the resources of untreated cytoplasm, while the rate of DNA synthesis of control nuclei decreased when they occupied cytoplasm which had been exposed to high doses of MNU. These studies suggest that nuclear sites of damage were only involved when lethal doses of MNU had been used.

Amoeba↗
PubMed1983

Quantitative analysis of changes in cell shape of Amoeba proteus during locomotion and upon responses to salt stimuli.

A new parameter expressing the complexity of cell shape defined as (periphery)2/(area) in 2D projection was found useful for a quantitative analysis of changes in the cell shape of Amoeba proteus and potentially of any amoeboid cells. During locomotion the complexity and the motive force of the protoplasmic streaming in amoeba varied periodically, and the Fourier analysis of the two showed a similar pattern in the power spectrum, giving a rather broad peak at about 2.5 X 10(-3) Hz. The complexity increased mainly due to elongation of the cell as external Ca2+ increased. This effect was blocked by La3+, half the inhibition being attained at 1/200 amount of the coexisting Ca2+. On the other hand, the complexity decreased due to rounding up of the cell as the concentration of other cations, such as Sr2+, Mg2+, Co2+, Ni2+, Na+, K+ etc., increased. Irrespective of the opposite effects of Ca2+ and other cations on the cell shape, the ATP concentration in amoeba decreased in both cases with increase of all these cations. The irregularity in amoeboid motility is discussed in terms of a dynamic system theory.

Adenosine Triphosphate↗
PubMed1992

Direct membrane effects of morphine and endorphins on Amoeba proteus.

Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1-8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.

Amoeba↗
PubMed2005

The testate lobose amoebae (order Arcellinida Kent, 1880) finally find their home within Amoebozoa.

Testate lobose amoebae (order Arcellinida Kent, 1880) are common in all aquatic and terrestrial habitats, yet they are one of the last higher taxa of unicellular eukaryotes that has not found its place in the tree of life. The morphological approach did not allow to ascertain the evolutionary origin of the group or to prove its monophyly. To solve these challenging problems, we analyzed partial small-subunit ribosomal RNA (SSU rRNA) genes of seven testate lobose amoebae from two out of the three suborders and seven out of the 13 families belonging to the Arcellinida. Our data support the monophyly of the order and clearly establish its position among Amoebozoa, as a sister-group to the clade comprising families Amoebidae and Hartmannellidae. Complete SSU rRNA gene sequences from two species and a partial actin sequence from one species confirm this position. Our phylogenetic analyses including representatives of all sequenced lineages of lobose amoebae suggest that a rigid test appeared only once during the evolution of the Amoebozoa, and allow reinterpretation of some morphological characters used in the systematics of Arcellinida.

Amoeba↗
PubMed2001

Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa.

Two cases of legionellosis occurring 3 years apart were acquired in the same French thermal spa and were apparently due to the same strain of Legionella pneumophila serogroup 1, as shown by genomic macrorestriction analysis. Minor differences between the two isolates were found by random amplification PCR profiling which showed an additional band with one of the isolates. Analysis of 107 L. pneumophila strains isolated from the spa waters by genome macrorestriction failed to identify the infective strain, but a closely related L. pneumophila serogroup 3 strain differing from the clinical isolates by only one band was found. To determine if the clinical L. pneumophila serogroup 1 isolates was better adapted for intracellular multiplication than related serogroup 3 environmental isolates, the growth kinetics of six isolates were determined in co-culture with Acanthamoeba lenticulata. One clinical isolate failed to grow within amoeba, while the other clinical isolate yielded the highest increase in bacterial cell count per amoeba (1,200%) and the environmental isolates gave intermediate values. Genetic analysis of L. pneumophila isolates by DNA macrorestriction does not therefore appear to reflect their growth kinetics within amoeba, and is not sufficiently discriminatory to identify potentially virulent strains.

Amoeba↗
PubMed1979

Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus.

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.

Amoeba↗
PubMed1996

Quantitation of free-living amoebae and bacterial populations in eyewash stations relative to flushing frequency.

This study investigated the concentration of amoebic and bacterial populations in eyewash station water relative to various flushing regimens. Amoebae concentrations averaged approximately 200 amoebae/100 mL in 13 of 15 stations positive for amoebae and consisted of Hartmannella and Acanthamoeba. Bacterial concentrations ranged from 10(0) to more than 10(5) colony forming units per mL. Amoebic concentrations differed notably between stations located in Buildings X and Y (p < 0.0001). Further study indicated that removal of diffusing screens did not substantially change (p > 0.05) the concentration of amoeba. Amoebic and bacterial concentrations temporarily decreased with the various flushing regimens tested. Lower amoebic concentrations were not sustained by a weekly 3-minute or a monthly 1-minute flushing regimen. However, weekly 3-minute flushes appeared to be more effective in maintaining lowered bacterial concentrations (p < 0.0001).

Amoeba↗
PubMed1957

Cytoplasmic incorporation of a ribonucleic acid precursor in Amoeba proteus.

The question of RNA synthesis in enucleate cytoplasm of Amoeba has been approached experimentally by incubating enucleate amoebae in a labelled RNA precursor and determining the incorporation into RNA autoradiographically. The results indicate that there is a cytoplasmic incorporation mechanism which can operate in the absence of the nucleus. A comparison is made between Acetabularia and Amoeba with respect to the origins of cytoplasmic RNA. It is concluded that the existing data are consistent with the assumption that some cytoplasmic RNA is of nuclear origin in both organisms.

Adenine↗
PubMed1979

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium.

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.

Amoeba↗
PubMed2002

Patients in the intensive care unit are exposed to amoeba-associated pathogens.

OBJECTIVE: To study the role of amoeba-associated alpha Proteobacteria as infecting agents in intensive care units (ICUs). DESIGN: Amoeba-associated alpha Proteobacteria were isolated from water samples taken from ICU taps and general hospital reservoir tanks using an amoebal co-culture procedure. Isolates were identified by 16S rDNA gene sequence comparison, and one isolate of each species was used as an antigen in a microimmunofluorescence assay to test the sera of the patients in the ICUs and compare them with those of control subjects. SETTING: The four university hospitals in Marseilles, France. PATIENTS: The sera of 85 patients in the ICUs with nosocomial pneumonia were tested. RESULTS: We isolated 64 bacterial strains that were identified as Afipia species or close relatives within the Rhizobiaceae subgroup of alpha Proteobacteria. These bacteria were assigned to 8 different species. Eleven patients were found to have an elevated antibody titer to either Afipia genospecies 1, or 3 still unnamed bacteria. No specific antibodies were detected in 100 control subjects (P < .01). CONCLUSION: These preliminary results support the hypothesis that ICU patients are exposed to amoeba-associated alpha Proteobacteria.

Alphaproteobacteria↗
PubMed2004

Amoebae in domestic water systems: resistance to disinfection treatments and implication in Legionella persistence.

AIMS: Monitoring of microbial changes during and after application of various disinfection treatments in a model domestic water system. METHODS AND RESULTS: A pilot-scale domestic water system consisting of seven galvanized steel re-circulation loops and copper dead legs was constructed. Culture techniques, confocal laser scanning microscopy after fluorescent in situ hybridization and viability staining with the BacLight LIVE/DEAD kit were used for planktonic and biofilm flora monitoring. Before starting the treatments, the system was highly contaminated with Legionella pneumophila and biofilm populations mainly consisted of beta-proteobacteria. In the water and the biofilm of the loops, continuous application of chlorine dioxide (0.5 mg l(-1)), or chlorine (2.5 mg l(-1)) were very effective in reducing the microbial flora, including L. pneumophila. Heterotrophic bacteria, although strongly reduced, were still detectable after ozone application (0.5 mg l(-1)), whereas with monochloramine (0.5 mg l(-1)) and copper-silver ionization (0.8/0.02 mg l(-1)), the contamination remained significantly higher. Monochloramine and copper-silver did not remove the biofilm. During copper-silver application, Legionella re-growth was observed. Only chlorine dioxide led to detectable effects in the dead leg. Amoebae could not be eliminated, and after interrupting the treatments, L. pneumophila quickly recovered their initial levels, in all cases. CONCLUSIONS: Chlorine dioxide, applied as a continuous treatment, was identified in this study as the most efficient for controlling L. pneumophila in a domestic water system. Chlorine dioxide showed a longer residual activity, leading to improved performance in the dead leg. Amoebae resisted to all the treatments applied and probably acted as reservoirs for L. pneumophila, allowing a quick re-colonization of the system once the treatments were interrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: Control of microbial contamination requires maintenance of a constant disinfectant residual throughout the water system. Treatment strategies targeting free-living amoebae should lead to improved control of L. pneumophila. Such treatment strategies still have to be investigated.

Amoeba↗
PubMed1985

Vertical distribution of potentially pathogenic free-living amoebae in freshwater lakes.

The vertical distribution of thermotolerant (37 degrees C and 45 degrees C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic particulate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by particulates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P less than 0.005) numbers of FLA were isolated from two particulate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe2+ state was predominant. In this midsummer period, 13 Naegleria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.

Amoeba↗