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We demonstrated that alkaline phosphatase was localized on the cell membrane of Dictyostelium discoideum amebae and on isolated plasma membranes. The enzyme activity was specifically inhibited by 0.01 M KCN or cysteine. The same method could also be applied to baker's yeast and MDCK cells (dog kidney cells in vitro).
Cell surface levels of 3H-cAMP binding to Dictyostelium discoideum amebae are dramatically reduced when cells are preincubated with cAMP. This decrease in 3H-cAMP binding is shown to reflect a loss in the number of binding sites and not in any significant change in their affinity constants(s). cAMP-mediated loss of its binding sites requires the continued presence of the cyclic nucleotide and does not depend upon protein synthesis. Reapparition of sites, which occurs when cAMP is eliminated from the media, also does not depend upon protein synthesis. Experiments using metabolic inhibitors and heat-killed cells suggest that the loss of binding sites is a direct consequence of the formation of cAMP-binding protein complexes.
We have used monoclonal antibodies to detect developmentally regulated cell surface antigens on D. discoideum amebae. A study of an antigen detected using an antibody produced by a hybridoma line implicates a previously undescribed component in the process of cell aggregation. This antigen (consisting of a doublet of 69,000 and 73,000 molecular weight) is first detected during the early hours of cell starvation and is present until cells begin slug formation. The developmental appearance of the antigen is not controlled by cAMP pulses and is distinct from that of Contact A sites. Fab fragments directed against the antigen are potent inhibitors of aggregation but do not inhibit the differentiation of cells to aggregation competence.
PURPOSE: To report a case of severe amebic keratitis in which both Hartmannella and Acanthamoeba were isolated simultaneously from the same lesion. METHOD: Case report. The deep corneal lesion was scraped for cytopathology and isolation of the pathogens. We tested the in vitro sensitivities of the pathogens to several drugs. RESULTS: Cultures of the corneal scrapings and of the solution in the patient's contact lens storage case were positive for Acanthamoeba E9 cysts and trophozoites. Hartmannella ameba coexisted with Acanthamoeba in the cornea. When tested in vitro, Acanthamoeba trophozoites were sensitive to both miconazole nitrate and natamycin, while cysts were sensitive only to natamycin. However, the patient did not respond to these antiamebic drugs. CONCLUSIONS: This case suggests that Acanthamoeba is not the only origin of amebic keratitis. Hartmannella may also cause severe drug-resistant keratitis.
One new and one known isoflavan, 3 S(+)-7-methoxymanuifolin K (1) and manuifolin K (2), respectively, were isolated from methanolic extracts of Dalea aurea (Fabaceae). Isoflavans 1 and 2 exhibited significant in vitro activity against the ameba Naegleria fowleri, an organism responsible for an infrequent but rapidly fatal form of primary amebic meningoencephalitis (PAM). At concentrations of 30 microM, both 1 and 2 caused growth inhibition of N. fowleri at a level comparable to amphotericin B (at 0.1 microM), the currently preferred treatment for this disease. Over a seven-day growth period, 1 and 2 (30 microM) exhibited superior growth inhibition of N. fowleri than amphotericin B after day 4. Isoflavan 2 was evaluated in a mouse model of PAM at a dose of 25 mg/kg/day for five days. While amphotericin B (2.5 mg/kg/day) offered 12.5 % protection of the mice, compound 2 did not protect the mice from PAM infection compared to controls.
In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier proposals based on the in vitro properties of these two proteins (Condeelis, J., M. Vahey, J. M. Carboni, J. DeMey, S. Ogihara, 1984, J. Cell Biol., 99:119s-126s).
The peripheral feeding network of the giant freshwater ameba Reticulomyxa can be easily and rapidly lysed to produce an extensive, stable, and completely exposed cytoskeletal framework of colinear microtubules and microfilaments. Most of the organelles that remain attached to this framework resume rapid saltatory movements at rates of up to 20 micron/s if ATP is added. This lysed model system is also capable of other forms of motility, namely an active splaying of microtubule bundles and bulk streaming. Reactivation does not occur with other nucleoside triphosphates, requires Mg ions, is insensitive to even high concentrations of erythro-9-(3-[2-hydroxynonyl]) adenine, is sensitive to vanadate only at concentrations of approximately 100 microM, and is inhibited by N-ethylmaleimide at concentrations greater than 100 microM. The physiology of this reactivation suggests an organelle transport motor distinct from cytoplasmic dynein and possibly the recently described kinesin. This system can serve as a model for elucidating the mechanisms of intracellular transport and, in addition, provides a unique opportunity to examine associations between microtubules and microfilaments.
Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.
Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.
The mitotic apparatus (MA) of the giant ameba, Chaos carolinensis, has characteristic sequences of microtubule arrays and deployment of nuclear envelope fragments. If mitotic organisms are subjected to 2 degrees C for 5 min, the MA microtubules are completely degraded, and the envelope fragments are released from the chromosomes which remain condensed but lose their metaphase-plate orientation. On warming, microtubules reform but show partial loss of their parallel alignment; displacement of the envelope fragments persists or is increased by microtubule reformation. This study demonstrates that cooling causes destruction of microtubules and intermicrotubular cross-bonds and further shows that such controlled dissolution and reformation can provide an in vivo test sequence for studies on the effects of inhibitor-compounds on microtubule subunit aggregation. Urea, at the comparatively low concentration of 0.8 M, inhibited reformation following cooling and rewarming but was ineffective in altering microtubules that had formed before treatment.
Amoeba proteus, Amoeba dubia, and Chaos chaos all contain similar plate-like and bipyramidal cytoplasmic crystals. Isolated crystals, purified by recrystallization from water, yield plate-like crystals which have been shown to be identical with synthesized carbonyl diurea (triuret) with regard to physicochemical properties, elemental analysis, x-ray diffraction patterns, infrared spectra, and optical properties. The birefringent plates found in the cytoplasm are carbonyl diurea. While the exact composition of the isotropic plates and of the bipyramids is not clear at present, the major constituent of these crystal types is also carbonyl diurea. It is suggested that carbonyl diurea is a nitrogen excretion product, not previously found in living organisms, and probably represents an end product of purine metabolism in amebae.
Changes in pH are measured in pinosomes and phagosomes of single specimens of the giant, free-living ameba, Chaos carolinensis. Measurements of pH are made microfluorometrically, as previously described (Heiple and Taylor. 1980. J. Cell Biol. 86:885-890.) by quantitation of fluorescence intensity ratios (Ex489nm,/Ex452nm, Em520-560nm from ingested fluorescein thiocarbamyl (FTC)-ovalbumin. After 1 h of pinocytosis (induced in acid solution), FTC-ovalbumin is found in predominantly small ( less than or equal to 5 micrometers in diameter), acidic (pH less than or equal to 5.0-6.2) vesicles of various shape and density. As the length of ingestion time increases (up to 24 h), the probe is also found in vesicles of increasing size (up to 100 micrometers in diameter), increasing pH (up to pH approximately 8.0), and decreasing density. Co-localization of fluorescein and rhodamine fluorescence, after a pulse-chase with fluorescein- and rhodamine-labeled ovalbumin, suggests vesicle growth, in part, by fusion. The pH in a single phagosome is followed after ingestion of ciliates in neutral solutions of FTC-ovalbumin. A dramatic acidification (delta pH greater than or equal to - 2.0) begins within 5 min of phagosome formation and appears to be complete in approximately 20 min. Phagosomal pH then slowly recovers to more neutral values over the next 2 h. pH changes observed in more mature populations of pinosomes within a single cell may reflect those occurring within a single phagosome. Phagosomal and pinosomal pH changes may be required for lysosomal fusion and may be involved in regulation of lysosomal enzyme activity.
Ionic relationships in the giant ameba Chaos chaos were studied by analyzing bulk preparations of ground cytoplasm for K, Na, and Cl. Ion levels under normal conditions were compared with the levels in cells exposed to varying concentrations of different ions, for varying times and at different temperatures. By standard intracellular electrode techniques, the bioelectric potential, electrical resistance, and rectifying properties of the plasmalemma were studied on intact cells in media of different composition. The results obtained, when related to evidence from other studies on ion fluxes and osmotic relationships, suggest the following concept of ionic regulation in Chaos chaos. In the absence of active membrane uptake, the plasmalemma is essentially impermeable to anions but permeable to both K and Na, which enter passively. In the cold the cell does not discriminate between K and Na, the cytoplasmic level of K + Na is determined by a Donnan distribution, and osmotic imbalance leads to slow swelling. At normal temperatures active processes are added: Na and water are pumped out by the contractile vacuole system; Cl is accumulated, along with the colloid components of the cytoplasm, only during feeding and growth, which depend upon membrane uptake and intracellular membrane transformations. There is no evidence for active transport of any ion species directly across the plasmalemma.
This report describes the use of the immunoenzymatic (peroxidase) method to identify the species and to stain distinctively the amebas in formalin-fixed paraffin-mounted sections. This permits the use of hematoxylin and eosin counterstaining. The method, now well developed by others for many purposes, is an alternative to immunofluorescence and seems to offer a number of advantages and a lesser number of disadvantages.
Comparative studies were conducted on the structure, nutrition, protein composition, immunology, and effect on cell cultures of Acanthamoeba sp. (Lilly A-1 strain), A. castellanii (Singh and Neff strains), A. astronyxis, A. comandoni, A. polyphaga, A. terricola, Hartmannella vermiformis, and Naegleria gruberi. Lilly A-1 strain of Acanthamoeba received special attention owing to its pathogenicity for experimental animals. Distinct differences were noted in structure, nutrition, and antigenic composition of Acanthamoeba spp. and Hartmannella, and it was concluded that their recognition as separate genera is justified. With the exception of A. terricola, all species of Acanthamoeba could be differentiated by cyst structure. Cysts of A. terricola closely resembled those of A. castellanii Singh strain, and close antigenic relationships between these 2 species were demonstrated by gel diffusion and immunoelectrophoresis (IEP); it was concluded that the 2 amebae belong in the same species. The pathogenic Acanthamoeba sp, Lilly strain differed from the nonpathogenic A. castellanii Singh strain (a) cyst structure; (b) protein distribution patterns (on disc electrophoresis);(c)soluble and particulate antigens (on gel diffusion, IEP, complement fixation, and immobilization tests); (d) capacity to induce cell-free plaques and other cytopathic effect (CPE) in mammalian monlayer cell cultures; (e) elimination of a phospholipase, responsible for some of the CPE, into the culture medium. Acanthamoeba sp. Lilly strain, which liberated more phospholipase, produced more CPE. Acanthamoeba sp. Lilly strain differed also from other species of this genus in cyst structure and antigenic composition. It was concluded, therefore, that, following the recommendation of Singh & Das, it ought to be placed in a separate species, Acanthamoeba culbertsoni.
Cell coats were cytochemically demonstrated for the first time in myxamebae of Fulig- septica, Didymium iridis, Dictyostelium discoideum, Cavostelium apophysatum, and amebae of Naegleria grubei. The stain enhances the cell coats of Physarum polycephalum plasmodia, Ceratiomyxa fruticulosa myxamebae, and Acanthamoeba sp. Cell coats usually unstained by cationic dyes stain intensely with the aid of the new cytochemical protocol utilizing 0.5% Alcian blue in the primary fixative and 0.05% ruthenium red in the secondary fixative.
Methods have beeni developed for controlling the transformation of Tetramitus rostratus, a protozoan able to reproduce either as an ameba or as a flagellate. An essential conidition for transformation is a low concentration of oxygen in the environment.
OBJECTIVE: The purpose of this report is to describe the clinical history, treatment, pathology, and imaging in two cases of rare CNS infection caused by free-living amebas. The Naegleria fowleri and Acanthamoeba species cause primary amebic meningoencephalitis and granulomatous amebic encephalitis, respectively. We describe the neuroimaging findings of a case involving a nonspecific cerebral edema pattern in primary amebic meningoencephalitis and a case involving focal enhancing lesions in granulomatous amebic encephalitis. CONCLUSION: Primary amebic meningoencephalitis and granulomatous amebic encephalitis have a grave prognosis and, although rare, should be considered in the differential diagnosis for patients who present with appropriate histories and imaging findings, including nonspecific brain edema on CT in primary amebic meningoencephalitis and focal punctate enhancing lesions in the posterior cranial fossa on T1-weighted MR imaging in granulomatous amebic encephalitis.