Phylogeny of the genera Entamoeba and Endolimax as deduced from small-subunit ribosomal RNA sequences

We sequenced small-subunit ribosomal RNA genes (16S-like rDNAs) of 10 species belonging to the genera Entamoeba and Endolimax. This study was undertaken to (1) resolve the relationships among the major lineages of Entamoeba previously identified by riboprinting; (2) examine the validity of grouping the genera Entamoeba and Endolimax in the same family, the Entamoebidae; and (3) examine how different models of nucleotide evolution influence the position of Entamoeba in eukaryotic phylogenetic reconstructions. The results obtained with distance, parsimony, and maximum-likelihood analyses support monophyly of the genus Entamoeba and are largely in accord with riboprinting results. Species of Entamoeba producing cysts with the same number of nuclei from monophyletic groups. The most basal Entamoeba species are those that produce cysts with eight nuclei, while the group producing four-nucleated cysts is most derived. Most phylogenetic reconstructions support monophyly of the Entamoebidae. In maximum-likelihood and parsimony analyses, Endolimax is a sister taxon to Entamoeba, while in some distance analyses, it represents a separate lineage. The secondary loss of mitochondria and other organelles from these genera is confirmed by their relatively late divergence in eukaryotic 16S-like rDNA phylogenies. Finally, we show that the positions of some (fast-evolving) eukaryotic lineages are uncertain in trees constructed with models that make corrections for among-site rate variation.     

Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting

ABSTRACT. Eighty-seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty-four distinct patterns were obtained, most of which corresponded to previously described species. In three species ( Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii ) intraspecific variation was detected that led to the grouping of isolates into 'ribodemes' (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis , which does not encyst, clusters with the third group indicating secondary loss of this ability.     

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy

Examination of Giardia muris cysts stained with the fluorogenic dyes, fluorescein diacetate (FDA) or propidium iodide (PI), by either Nomarski differential interference contrast (DIC), phase, or brightfield (BF) microscopy revealed a direct correlation between morphologic appearance and uptake of FDA or PI. Cysts incorporating FDA were all morphologically identical and exhibited (1) a clearly delineated cyst wall, (2) the presence of a distinct space between cyst wall and cytoplasm, and (3) flagella recognizable at one pole of the cyst. FDA-positive cysts also had a hyaline appearance of the cytoplasm (examined at multiple focal planes with DIC) that made it very difficult to detect the presence of nuclei, intracellular axonemes of flagella, or curved elements of the adhesive disc. However, PI-stained cysts possessed a distinct morphology that was clearly different from that of FDA-stained cysts. Examination of PI-stained cysts demonstrated the presence of well-defined nuclei, intracellular axonemes, and curved elements of the adhesive disc. The cytoplasm of PI-stained cysts contained a fine granular texture as opposed to the hyaline appearance of FDA-stained cysts, and no space was observed separating the cyst wall from the underlying cytoplasm in the PI cyst. This light microscopic comparison of viable FDA- and nonviable PI-stained cysts of G. muris demonstrates that 2 types of cysts can be distinguished and implies that structural differences can be used to identify these subpopulations of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

转GFP::cDNA基因拟南芥筛选及相关基因的功能分析

本研究以随机GFP::cDNA融合基因转基因拟南芥为材料,筛选到在细胞核或在细胞核和细胞质中均表达GFP信号的转基因株系58个。对这些转基因株系中的cDNA插入片段进行克隆,获得4株插入片段能按原初编码框进行编码的转基因株系。对插入片段为编码富含甘氨酸蛋白AtGRP8 C-末端(富含甘氨酸的结构域)的转基因株系R2的表型分析发现,连续白光、红光或蓝光下其幼苗的下胚轴比野生型的要短,且较低光照强度白光(低于100μmol m-2s-1)、蓝光(低于75μmol m-2s-1)下的差异更加明显,但是黑暗中其幼苗的下胚轴与野生型相比无明显差异,表明AtGRP8蛋白可能通过其C-末端功能域参与调控拟南芥的光形态建成反应。     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Modifications of Kohn's chlorazol black E staining and Wheatley's trichrome staining for temporary wet mount and permanent preparation of Entamoeba histolytica.

Preparation of stained smears of Entamoeba histolytica has several drawbacks. We therefore tried to simplify the staining procedures by modifing Kohn's chlorazol black E staining and Wheatley's trichrome staining techniques. Trophozoites and cysts of axenically cultured E. histolytica and Entamoeba invadens, respectively, and trophozoites and cysts of E. histolytica in stools of patients were used. Karyosomes and peripheral chromatin of nuclei and chromatoid bodies became distinctly visible after amoebae were suspended in the basic solution of Kohn's stain. Amoebae fixed in suspension with either basic solution or Bouin's fixative were clearly stained with Kohn's and trichrome preparations, both as wet mounts directly and as permanent slides after processing for mounting. These procedures were easier when the basic solution was used as a fixative and trichrome stain was employed. Erythrocytes ingested by trophozoites, however, were not stained with either of these preparations after fixation in the basic solution but were clearly stained when Bouin's fixative was used. Cysts of E. histolytica in stools concentrated using basic solution (instead of formalin) and ether were also stained with these stains. Consequently, without employing highly toxic mercuric chloride, wet mounts and permanent smears can be prepared with permanent stains, and preserved cysts can be stained after concentration.     

Amoebiasis: diagnosis by aspiration and exfoliative cytology

The present study was undertaken to evaluate the use of fine needle aspiration and exfoliative cytology in the identification of amoebic cysts/trophozoites, and to characterize amoebiasis. The subjects consisted of 15 patients, 11 diagnosed by fine needle aspiration cytology (FNAC) as amoebic abscesses (14 liver and one pulmonary) and four women whose cervical smears contained Entamoeba histolytica cysts or trophozoites. Of 128 ultrasonographically guided FNAC of hepatic lesions over a four year period, 17 were abscesses of which 10 were diagnosed as amoebic. A single case of pulmonary amoebiasis was detected in an 18-year-old male. The case was initially diagnosed as tubercular due to deceptive symptomatology. Three cases of amoebic cysts and one trophozoite were reported on routine cervical smear screening. All four cases were unsuspected for amoebic infection. The disease may easily go undetected unless meticulous screening is exercised, and the search for cysts or trophozoites is made with clear concepts of the morphological characteristics of E. histolytica in mind.     

A Hyperparasite of Entamoeba suis from the Indian Domestic Pig Sus scrofa*

SYNOPSIS. A hyperparasite probably related to some lower protistan was found in the cytoplasm of Entamoeba suis Hartmann. These were very minute bodies with a central Feulgen-positive dot, the nucleus, and a bordering cytoplasmic ring with an alcian blue-positive reaction. This organism appeared to interfere with the synthesis of DNA & RNA in the host amoeba as reflected in its relative stainability with Feulgen or pyronin-methyl green. The suggestion is that the parasitized amoebic cysts were most likely rendered nonviable.     

Two species of Entamoeba from white-tailed deer, Odocoileus virginianus, from Georgia

Large numbers of 2 species of Entamoeba, Entamoeba coli and Entamoeba bovis, were recovered from captive white-tailed deer from Georgia. The trophozoite of the first ameba was a large form with a well-defined vesicular nucleus bearing a prominent eccentric endosome, abundant peripheral chromatin, and numerous periendosomal granules. Cytoplasmic vacuoles containing bacteria and other substances were common. Mature cysts were octonucleate. Glycogen vacuoles and irregularly shaped chromatoids were frequently present. This ameba was identified as Entamoeba coli. The trophozoite of the second ameba was small, with a typical Entamoeba nucleus. Cysts were uninucleate, containing numerous chromatoids resembling bars, splinters of wood, and irregular masses. There was occasionally a glycogen vacuole. This ameba was identified as Entamoeba bovis.     

Numbers of muscle nuclei and myosatellite cell nuclei in red and white axial muscle during growth of the carp (Cyprinus carpio)

We determined the percentages of muscle fibie nuclei and satellite nuclei over a growth range of carp ( Cyprinus carpio ), as the increase in the number of muscle fibre nuclei is an important aspect of the increase in muscle mass, and myosatellite cells are believed to be the source of new muscle fibre nuclei. In white as well as in red axial muscle the percentage of the nuclei present in muscle that are muscle nuclei (muscle fibre nuclei+myosatellite nuclei) remained constant during growth (54 and 32% respectively). The difference in the percentage of non-muscle nuclei between white and red axial muscle is mainly caused by the higher content of endothelial nuclei in red axial muscle.
In white axial muscle the DNA/protein ratio (nucleus/sarcoplasm ratio) decreased between 3 and 15 cm S.l. In red axial muscle we found a continuous decrease in DNA/protein ratio over the entire investigated size range (3–50 cm s.l.). This may be related to a longer occurrence of hyperplasia in red than in white axial muscle.
In both fibre types the percentage of muscle nuclei being myosatellite nuclei decreased with increasing length, In white axial muscle it decreased from about 5% in carp of 5 cm s.l. to less than 1% in carp of 20 cm S.L.; for red muscle these values were 11 and 3% respectively.
For white axial muscle we calculated that, especially in larger fish, the myosatellite ceils alone cannot account for the increase in the number of muscle fibre nuclei during growth. The percentage of proliferating nuclei in muscle tissue, measured by the uptake of 5-bromo-2'-deoxy-uridine, is high enough to account for the total increase in nuclei. So indirect evidence is available that another cell type present in the muscle tissue may also be involved in the formation of additional muscle fibre nuclei.     

Impact of solar radiation in disinfecting drinking water contaminated with Giardia duodenalis and Entamoeba histolytica/dispar at a point-of-use water treatment

Aims:  To determine the impact of natural sunlight in disinfecting water contaminated with cysts of Giardia duodenalis and Entamoeba histolytica/dispar using plastic containers.
Methods and Results:  Known quantities of Giardia duodenalis and Entamoeba histolytica/dispar cysts in sterile water were exposed to the sun. Containers were made of polyethylene terephthalate, eight painted black on one side, one not painted and another cut open at the top and the last was a high density polypropylene container. Viability testing was performed using vital and fluorescent dyes. The same assays were conducted under cloudy conditions. Thermal control tests were also performed using heat without ultra violet light from the sun. Results show that 99·9% of parasites was inactivated when water temperatures reached 56°C after sunlight exposure.
Conclusion:  Both solar radiation and heat produced by the sun have a synergistic effect in killing cysts of Giardia duodenalis and Entamoeba histolytica/dispar when temperatures rise above 50°C, with complete death at 56°C, using painted 2-l PET containers.
Significance and Impact of the Study:  Solar disinfection system using PET containers painted black on one side can be used to disinfect water against Giardia duodenalis and Entamoeba histolytica/dispar using natural sunlight.     

Ribonucleoprotein-Containing Vesicles in Cysts of Naegleria gruberi*

SYNOPSIS. Ultraviolet microscopy and electron microscope autoradiography were used to study ribonucleoprotein in cysts of Naegleria gruberi. The absorption maximum for cysts is at 265 nm with little detectable absorption occurring at 295 nm. Pre-cystic trophozoites absorb less strongly than the cysts at 265 mm. Acridine orange staining indicated concentrations of ribose nucleic acid or ribonucleoprotein in the cytoplasm of young cysts. The dye stained discrete vesicles in the cytoplasm. Tritiated uridine and tritiated proline were used to follow changes in RN-protein at encystment. Label was incorporated into vesicles filled with ribosome-like particles. These are presumably the sites of acridine orange staining. Relatively little label was associated with the cyst cytoplasmic matrix; most of the silver grains lay over the nucleus and cytoplasmic organelles. The vesicles are believed to represent autophagosomal-type vacuoles with the contents derived from breakdown of organelles such as mitochondria. The path of label into the vesicles is via lysis of labeled cytoplasmic organelles. The RN-protein vesicles of Naegleria gruberi cysts are compared to the chromatoid bodies of Entamoeba invadens. It is concluded that, tho differences in detail are present, the role of the structures in the cysts is probably the same. They are a ready source of amino acids and ribosomes in a dedifferentiated or pool state to be used for synthetic reactions that accompany resumption of trophic existence.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

8-Oxoguanosine and uracil repair of nuclear and mitochondrial DNA in red and white skeletal muscle of exercise-trained old rats.

Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.     

Tumor Cells Genetically Labeled with GFP in the Nucleus and RFP in the Cytoplasm for Imaging Cellular Dynamics

Dual-color fluorescent cells with one color fluorescent protein in the nucleus and another color fluorescent protein in the cytoplasm were genetically engineered. The dual-color cancer cells enable real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of a series of human and rodent cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Using the Olympus OV100 Whole-Mouse Imaging System, total sub-cellular dynamics can be visualized in the living dual-color cells in real time in the live mouse after cell injection. Highly elongated cancer cells and nuclei in narrow capillaries were visualized where both the nuclei and cytoplasm deform. Both cytoplasm and nuclei were visualized to undergo extreme deformation during extravasation with cytoplasmic processing exiting vessels first and nuclei following along these processes. The dual-color cells described here thus enable the sub-cellular dynamics of cancer cell trafficking to be imaged in the living animal.     

Entamoeba bubalus, n.sp., from Carabao

SUMMARY. Twelve carabao in the Philippines were found to be lightly infected with intestinal amoebae. Trophic forms (12 microns in diameter) of this protozoan possessed a definite ectoplasm and a homogeneous endoplasm. They were found only in stained preparations. The nucleus was similar to that of the cyst. All unconcentrated fecal smears contained at least a few cysts (8 microns in diameter). In these forms the cytoplasm usually contained a large vacuole and one or more irregular chromatoidal bodies. The nucleus (2.6 microns in diameter) possessed a pronounced, deeply staining, uniform peripheral ring and a large irregular endosome. There was no periendosomal ring. This amoeba is designated as Entamoeba bubalus , n.sp.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

ULTRASTRUCTURE OF GLOEODINIUM MONTANUM (DINOPHYCEAE)1

The freshwater dinoflagellate Gloeodinium montanum Klebs (1912) was examined with transmission and scanning electron microscopy. Micrographs of ultrathin sections revealed a series of membrane layers rather than the usual dinoflagellate theca in vegetative cysts and in legates. Swarmers had distinct pellicles but appeared to be devoid of thecal plates and vesicles. The organization of cysts and swarmers appeared remarkably similar. All cell types had typical dinoflagellate nuclei with condensed chromosomes. Chloraplasts had girdle lamellae. One pyrenoid per cell was also present in chloroplasts of vegetative cysts. Starch grains and oil globules were distributed throughout the cytoplasm. Large accumulation bodies and polyvesicular vacuoles were found in aging cysts. Trichocysts and flagellar hairs were absent. Two types of intra-cellular prokaryotic organisms were discovered.     

A new fluorescent test for cell vitality using calcofluor white M2R

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.