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PubMedDate not supplied

Characterization of a monoclonal antibody and a cDNA for polyubiquitin of Amoeba proteus.

A monoclonal antibody was obtained that reacts with many different proteins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross-reacted with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages. Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well-conserved 20Ser and 55Thr residues were replaced with Gly and Ser, respectively. The 28Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin-mRNAs that could be detected by Northern blot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiquitinated in response to cellular stresses.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.

Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.

Amino Acid Sequence↗
PubMed1998

[Isolation of Legionella and free-living amoebae at hot spring spas in Kanagawa, Japan].

Microbiological contamination of hot spring bath water is a public health concern. A province-wide survey was carried out to determine the extent and distribution of both Legionella and free-living amoebae contamination. Among 30 samples of hot spring bath from 12 sites in Kanagawa, Japan, L. pneumophila was detected in 21 water samples from 11 sites, ranging from 10(1)-10(3) CFU/100 ml. Serogroups 3, 5 and 6 of L. pneumophila were predominantly isolated from the samples. Naegleria (46.7%), Platyamoeba (33.3%), Acanthamoeba (10.0%) and 2 other genera of free-living amoebae were detected in 22 samples from 11 sites. One or more genera of host amoebae of Legionella occurred in 17 samples (56.7%) from 9 sites. Another thing to be noted is that 13 water samples contained N. lovaniensis. Although N. lovaniensis is nonpathogenic, it is considered an indicator organism for places that are suitable for the growth of N. fowleri, a causative agent of primary amoebic meningoencephalitis in man.

Amoeba↗
PubMed1984

Phenothiazine compounds inhibit in vitro growth of pathogenic free-living amoebae.

The phenothiazine compounds trifluoperazine dihydrochloride and chlorpromazine hydrochloride have in vitro activity against the pathogenic free-living amoebae Naegleria fowleri, Acanthamoeba culbertsoni, and Acanthamoeba polyphaga. Drug concentrations of 10 microM were amoebastatic; concentrations of 50 microM were either amoebastatic or amoebicidal. Concentrations of 100 microM were generally amoebicidal. The mechanism of drug action is unclear. It may reflect sensitivity of amoeba calcium regulatory protein to the phenothiazine compounds or may be due to the lipophilic action of the drugs on the amoeba plasma membrane. Accumulation of these drugs in the central nervous system makes them potentially useful chemotherapeutic agents in humans for treatment of amoebic meningoencephalitis caused by N. fowleri and Acanthamoeba spp.

Amebicides↗
PubMed1982

Isolation of amoebae from edible mushrooms.

Isolation of amoebae from the surface and internal tissues of edible mushrooms was investigated. Samples tested over a 3-year period included mushrooms cultivated from six geographic localities. Of 168 mushroom surfaces tested, 161 (96%) yielded amoebae. Of 166 samples of internal stalk and cap tissues tested, only 1 yielded amoebae.

Amoeba↗
PubMed1981

A fatal case of meningoencephalitis due to a free-living amoeba of uncertain identity--probably acanthamoeba sp.

There are 2 main types of meningoencephalitis caused by free-living amoebae. The first is a well-defined acutely fatal disease resembling fulminating bacterial meningitis. It is caused by the single species Naegleria fowleri. The second is a more poorly defined disease that runs a subacute or chronic course and is characterized by focal granulomatous lesions in the brain. The causative organisms are probably Acanthamoeba sp. in most cases, but it is possible that other genera may be involved. The first case of the subacute form of the disease to be recognized in Australia is described. A 2 1/2-yr-old, previously well girl presented with ataxia and lower motor neurone paralyses. The cerebrospinal fluid was pleocytic and she was thought to be suffering from a relatively minor viral brain-stem encephalitis. Her symptoms persisted in a peculiarly fluctuating way for 30 d when she suddenly collapsed and died from an intracranial haemorrhage. Necropsy showed focal granulomatous lesions associated with necrotizing vasculitis in the basal regions of the brain. The lesions contained well preserved free-living amoebae which were morphologically different from N. fowleri and most closely resembled Acanthamoeba sp. The ultrastructure of the organisms was particularly well preserved and is described in some detail. Immunohistological studies also excluded N. fowleri but were inconclusive for Acanthamoeba or other genera of free-living amoebae. Difficulties with the diagnosis and treatment of this disease are discussed and some practical suggestions are made.

Amoeba↗
PubMed1999

Epizoic amoebae from the gills of turbot Scophthalmus maximus.

Species of amoebae belonging to the genera Platyamoeba Page, 1969, Vannella Bovee, 1965 and Flabellula Schaeffer, 1926 were found to accompany Paramoeba sp., the agent of amoebic gill disease (AGD), in clinically diseased turbots. The same community of epizoic gymnamoebae was found on the gills of turbots which revealed no gill abnormalities but slight behavioral signs indicative of suboptimal health status. The assemblage of the above-mentioned free-living amoebae capable of colonizing gill tissue of turbots was supplemented with species recognized in samples fixed from primary isolates for transmission electron microscopy. The pathogenic potential of epizoic gill amoebae in turbots is discussed.

Amebiasis↗
PubMed1978

Meningoencephalitis and brain abscess due to a free-living amoeba.

A 47-year-old diabetic woman developed fatal meningoencephalitis due to a free-living amoeba. The responsible organism appeared to be neither Naegleria nor Acanthamoeba-Hartmannella. Both acute and chronic (granulomatous) inflammatory reactions of the brain were present, and both cysts and trophozoite forms were readily visualized. The latest classification and methods of identification of such amoebae are reviewed and their threat to public health underscored. It should be realized that a variety of free-living amoebae exist in nature, which potentially can produce meningoencephalitis in humans, and that none of these organisms should be labeled or considered as "avirulent" or "nonpathogenic" until proved otherwise.

Amebiasis↗
PubMed2000

[Amoeba of the Acanthamoeba species in swimming pools and lakes of the Lublin area and in people using contact lenses].

Examinations of water for the presence of Acanthamoeba in covered and open air swimming pools and lakes of the city of Lublin were performed in order to determine the places where the invasion of amoebas of Acanthamoeba kind on the eyes can occur. Pathogenic strains of Acanthamoeba were found in all collected water samples. The cultures from the conjunctival sac of regular swimmers practising every day in these pools were taken. Amoebas were found in none of the examined cultures. The observations have shown that contact lenses should be protected from the contamination with swimming pool water. Amoebas were not revealed in the conjunctival sac of the swimmers after bathing.

Adult↗
PubMed1975

On the nature of hyaline zones in the cytoplasm of Amoeba proteus.

Investigations with the Nomarski DIC (differential interferece contrast) microscope and the electron microscope on the nature of hyaline zones in the cytoplasm of Amoeba proteus revealed that these regions represent pure ground cytoplasm. Differences between specimens 1) treated with 2% ethanol, 2) released from high hydrostatic pressure or 3) preincubated at 35 degrees C for 30 minutes could not be observed. Only dying cells undergoing lysis contained a watery solution within the zones of hyaline appearance. The existence of a so-called cell surface complex composed of the plasma membrane and an electron dense filamentous layer of groundplasm was demonstrated by the electron microscopical analysis of narcotized and pre-heated amoebae. This complex corresponds morphologically to the cell surface complexes in tissue cells. Hence it seems possible that the cell surface complex of amoebae is also responsible for changes of the cell shape and movements of the cell membrane. Observations with the DIC microscope also revealed the existence of two types of hyaline caps in A. proteus: in pseudopodia extending during normal locomotion the hyaline cap consists of pure ground cytoplasm, whereas in specimens showing fountain-like streaming the cap is built up by a large vacuole containing a watery fluid.

Amoeba↗
PubMed2004

Bloody diarrhoea cases caused by Shigella and amoeba in Jordan.

This study was done to evaluate the occurrence of bloody diarrhoea in Jordan in the period 1988-2000, with particular interest in Shigilla and amoeba cases in relation to the surveillance control system and hygiene policy. The cases were collected from various medical diagnostic laboratories. In addition to the clinical presentation, stool specimens or rectal swabs were collected and sub-cultured on the appropriate media. Shigella spp. and amoeba were identified using standard methods. Anti-microbial susceptibility tests were performed. One-way ANOVA and Least Significant Difference (LSD) were used for statistical analysis. The number of stool specimens tested for bloody diarrhoea was 34,529 in the study period. Positive stool samples were reported in 711 (2.1%) patients. Out of these 55% were males and 45 % were females. The highest number of cases 96 (13.5%) was reported in the year 1999. The highest incidence rate was (20.6) in the year 1996 and the lowest incidence rate (8.9) was in the year 2000. The highest number of cases during the period 1998-1992 was among age group 1-4 year. During the year 1993-1996 the highest number of cases was among group 5-14 year. The highest number of cases during the year 1997-2000 was among age group 15-24 year. There was a significant variation between numbers of bloody diarrhoeal cases and age groups (P < 0.001) as well as sex (P = 0.028). No significant variation (P = 0.06) was observed between number of bloody diarrhoea cases and seasons or months. The number of stool specimens tested for amoebiasis during the study period 1994-2000 was 229,040. Shigella was isolated from 304(0.13%) cases and amoeba was found in 24,211(10.6%) cases. The lowest incidence rate in bloody diarrhoeal cases in the year 2000 indicates good quality hygienic control. The increased resistance of Shigella to antibiotics may produce a future problem, which might require a further prescribing policy for treatment.

Adolescent↗
PubMed2005

[A micro-culture method for continuous observation of free-living amoebae].

OBJECTIVE: To establish a method for co-culture of amoebae and endosymbionts, also for continuously observing the microphenotype of amoebae. METHODS: 24 wells culture plate with cover glass on the wells was used as containers. Amoebae and Candida albicans were co-cultured in microdrop of medium in the wells at 37 degrees C, and observed under x1000. RESULTS: Continuous observation revealed trophozoites in various shapes like letters T, K, or Y, their movement and ingestion phenomenon were observed. CONCLUSION: The micro-culture method is useful in observing the amoebal morphology and its phagocytic process to Candida albicans.

Amoeba↗
PubMed1991

Fresh-water amoebae from four aquatic sites in Egypt.

Water samples from four aquatic sites in Egypt were examined for the presence of free-living amoebae. Amoebae were isolated and cultured on 1.5% non-nutrient agar with bacteria. They were studied morphologically and tested for production of flagellated forms, encystment, excystment, nuclear division, temperature tolerance and pathogenicity in young mice (8 - 10 gm). Didascalus thorntoni and Naegleria gruberi were dominant in all surface waters. Naegleria fowleri, Acanthamoeba glebae, A. culbertsoni and A. rhysodes were found in Lower Egypt, while Vahlkampfia avara and V. thornata were found in Upper Egypt. All species tolerated incubation temperatures of up to 36 degrees C except for A. rhysodes which degenerated at 34 degrees C. A. culbertsoni tolerated 38 degrees C but died at 40 degrees C. Amoebae were detected histologically in a necrotic brain tissue from only one mouse which was inoculated with A. culbertsoni.

Amebiasis↗
PubMed1990

Glutathione transferase activity in some flagellates and amoebae, and purification of the soluble glutathione transferases from Acanthamoeba.

Glutathione transferase (GST) enzymes are toxicologically important from many points of view. Nine protozoans were investigated here for their GST content. Six aerobic amoebae had very different specific GST activities, but an anaerobic amoeba and two anaerobic flagellates did not have any GST activity, suggesting that the peroxidase activity of GST is an evolutionarily important property for aerobic organisms. The soluble GST isoenzymes of Acanthamoeba culbertsoni and A. polyphaga were purified and partially characterized. The same two cationic and one anionic GST isoenzyme were found in both Acanthamoeba ssp., while A. culbertsoni had one more cationic isoenzyme. It is concluded that GST in aerobic amoebae can play an important role in detoxication.

Acanthamoeba↗
PubMed1978

A capillary technique for cloning Amoeba from single cells.

Each amoeba is placed in a drop of approximately 40 microliter of a balanced 'medium + food' solution introduced into the central region of a 7 cm long capillary tube which has first been fused at one end. Such amoebae divide to form clones of 20-30 cells without changing capillaries, though additional food may be required after 7-10 days. Experiments on damage induced by chemicals, cell cycle sensitivities and nuclear/cytoplasmic inheritance, which may involve the cloning of some 2-3,000 or more cells can be carried out with comparative ease. Apart from research, the capillary cloning technique has proved useful in the teaching laboratory. It is adaptable, and can be used for Protozoa other than amoebae, even for requiring sterile conditions.

Amoeba↗
PubMed1984

Human neutrophils require activation by mononuclear leucocyte conditioned medium to kill the pathogenic free-living amoeba, Naegleria fowleri.

Naegleria fowleri is a free-living amoeba which causes a fulminant and rapidly fatal meningoencephalitis in man. Human neutrophils fail to kill the amoeba in vitro, but can do so if they are exposed to conditioned medium (CM) from PHA stimulated mononuclear leucocytes (MNLs). Specific antibody or complement was required to effect amoeba killing by CM modified neutrophils. Only short time exposure of the leucocytes to CM was required to endow them with amoebicidal properties. The CM was also shown to contain neutrophil migration inhibition activity and an activity(ies) which induced a respiratory burst in neutrophils. The results highlight the importance of MNL products other than specific antibody in neutrophil anti-microbial activity.

Amoeba↗
PubMed1984

[Observation of filaments of 10nm in the demembraned cytoplasm of Amoeba proteus].

We report here the first observation of 10 nm filaments in a protozoan, Amoeba proteus. These intermediate sized filaments were observed in spread cytoplasmic preparations of amoeba as stable cytoplasmic components over a wide range of pH (5.0-9.0). Although their morphology is grossly similar to the vertebrate intermediate filaments by negative staining, the filaments of amoeba show a characteristic helical structure with a 25 nm axial periodicity and do not display fibrillar projection along their length or at their extremity.

Amoeba↗
PubMedDate not supplied

Survey of fresh vegetables for nematodes, amoebae, and Salmonella.

Contamination by nematodes, amoebae, and bacteria of the genus Salmonella was estimated in a 2-year survey of salad vegetables obtained from wholesale and retail sources. The vegetables examined were cucumbers, cabbage, lettuce, celery, carrots, radishes, tomatoes, mushrooms, cauliflower, and spinach. Nematode eggs and larvae were recovered by the Nacconol-ether centrifugation method. Some nematode eggs were identified as parasitic Ascaris sp.; the majority of larval nematodes were thought to be soil-dwelling species. Amoebae were recovered by rinsing the vegetables with distilled water, centrifuging the rinse water, and transferring the sediment to agar plates on which a bacterial lawn had previously been grown; trophozoites identified as the potentially pathogenic species--Acanthamoeba polyphaga, A. rhysodes, and A. castellanii--were the most common amoebae recovered on the plates. Salmonella spp. were grown from 4 of 50 samples.

Amoeba↗