Morphology of the intranuclear inclusions in Entamoeba histolytica trophozoites

Electron microscope studies of spherical intranuclear inclusions (II) of Entamoeba histolytica trophozoites of two strains, which were maintained in polyxenous culture for different periods of time, were carried out. Data on the number of II in different individuals and in different strains are given. The structure of II whose external layer consists of electron dense material and resembles morphologically peripheral chromatin of the ameba's nucleus, an analogy of nucleolus chromatin of cells of other eucariots, is considered. Inside this layer some II have annular filaments about 9 nm thick rolled up spherically around the central zone of II. Comparison of results obtained and literary data suggests that the dynamic structure of II in question reflects a number of biosynthetic processes on the basis of annular extrachromosomal DNA of exogenic origin.     

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.     

Introns of Entamoeba histolytica and Entamoeba dispar.

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar, indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to the corresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences G     

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens

Abstract Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.     

Effects of amiloride on Na+ content and pinocytosis in Entamoeba histolytica

Amiloride, a blocker of Na+ leak and Na+-H+ exchange in animal cells, caused cells of Entamoeba histolytica to release Na+ (up to 40% of their original Na+ content within 90 min, at an amiloride concentration of 3 mM); K+ content was not affected. By comparing the unidirectional uptake of 22Na+ with that of the fluid-phase marker 125I-labeled poly(vinylpyrrolidone) we established that the amiloride-induced Na+ loss was due to inhibition of pinocytic Na+ uptake rather than to blockage of an amiloride-sensitive transport system in the plasma membrane. Amiloride penetrated the cells, and both its intracellular concentration and its effect on pinocytosis increased with pH. The permeant weak base quinacrine similarly inhibited pinocytosis in a pH-dependent manner. We conclude that the effect of amiloride on pinocytosis and, consequently, on Na+ content was due to its properties as a permeant weak base.     

Entamoeba histolytica: correlation between virulence and content of proteolytic enzymes

Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.     

Mechanism of L-serine oxidation in Entamoeba histolytica.

1. The enzymatic mechanism of oxygen uptake elicited by L-serine in axenically cultivated trophozoites of Entamoeba histolytica was investigated. 2. Of 22 amino acids examined, only L-serine stimulated oxygen consumption by intact and disrupted amoebae. 3. Pyruvate, a product of serine metabolism, also stimulated oxygen consumption in the amoebae. 4. Characterization of the oxygen uptake elicited by both L-serine and pyruvate, and analysis of the products of L-serine metabolism indicate that the amino acid is first converted to pyruvate. 5. L-Serine dehydratase, which catalyzes the deamination of serine to pyruvate, was detected primarily in the soluble fraction of the amoebae. D-Serine potently inhibited the enzyme, as well as oxygen uptake in the presence of L-serine but not in the presence of pyruvate. 6. The pyruvate formed is oxidized, at least in part, by a novel pyruvate oxidase involving the uptake of molecular oxygen.     

Inorganic pyrophosphatase and nucleoside diphosphatase in the parasitic protozoon, Entamoeba histolytica.

Entamoeba histolytica contains two acid pyrophosphatases. One is an inorganic pyrophosphatase with a relatively high K_m ( ? 1 mM) and no cation requirement. The other is a nucleoside diphosphatase with a relatively low K_m ( ? 50 μM) and Ca²⁺ requirement. No Mg²⁺ dependent neutral or alkaline inorganic pyrophosphatase is present. The pyrophosphatases are localized in subcellular particles, display structure-linked latency and are tightly bound to membranes.     

Effects of nitrogen and phosphorus starvation on nucleic acid and protein content of Heterocapsa sp.

Changes in the protein, RNA and DNA content related to nitrogen(N) and phosphorus (P) starvation were studied in the marinedinoflagellate Heterocapsa sp. grown in batch cultures. In bothcases of nutrient starvation, metabolic adaptations affectedprotein and RNA pools, while the DNA content per cell remainedapproximately constant. N starvation led to a parallel decreasein protein and RNA concentration which caused the protein/RNAratios to remain constant. A dramatic decrease in the RNA contentcharacterized the P-starved cultures, although protein synthesiscontinued. The ribosomal RNA content was lower than expectedgiven the continuation of protein synthesis. It is suggestedthat protein/RNA ratios could be used as an indicator of P starvation,while protein/chlorophyll ratios would characterize N starvation ¹Present address: University of Hawaii at Manoa Soest, BiologicalOceanography, 1000 Pope Road, Honolulu, HI 96822, USA     

Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.     

Physiological significance of glucose transport in Entamoeba histolytica.

A glucose transport system, previously found in a bacterial grown strain of Entamoeba histolytica, is also present in a strain grown in axenic culture and in an atypical strain which can grow at room temperature. The last strain has a lower temperature coefficient for glucose transport than the two typical strains, which grow only above 33 C. The uptake of glucose by pinocytosis is much lower than the uptake through the specific transport system. The rate of glucose transport was equal to the rate of glucose consumption from the medium. No free glucose could be detected inside amoebal cells incubated with external glucose. All these observations are consistent with the idea that transport is a rate limiting step in the utilization of glucose by E. histolytica.