Autoradiographic Studies of H3-Thymidine Uptake in Entamoeba histolytica in CLG Medium*

SYNOPSIS. Entamoeba histolytica grown with H³-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome. The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H³-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H³-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.     

Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Incorporation of glucose carbon into ribonucleotides of axenic Entamoeba histolytica

Axenic Entamoeba histolytica grown with (UL-14C)glucose in TP-S-1 medium were capable of biosynthesizing ribose from labeled glucose. RNA isolated by phenol extraction was hydrolyzed to the ribonucleotide level by alkaline hydrolysis. The hydrolysate, chromatographed on ion exchange resins, yielded AMP, GMP, and UMP, but not CMP containing labeled glucose carbon. The present nucleotide composition of the isolated amebal RNA was, respectively, as follows, CMP, 0.20; GMP, 0.22; AMP, 0.30; UMP, 0.29. The location of all the radiolabel in each ribonucleotide was the ribose moiety. The relative specific incorporation of glucose carbon into AMP, GMP, and UMP was 0.47, 0.05, and 0.10, respectively. These results suggest that the bulk of amebal nucleic acid precursors are obtained as preformed nucleosides and/or nucleotides from TP-S-1 medium. The mean RNA content per milliliter packed cells of amebae was 4.2 +/- 0.2 mg.     

Autoradiographic Studies of Uridine-5-H3 Uptake in Entamoeba histolytica in the CLG Medium1

SYNOPSIS. Using uridine-5-H³, “long-term” labeling experiments over a 72 hr growth cycle were done with E. histolytica strain K9 grown in CLG medium with penicillin-inhibited Bacteroides. Autoradiographic analysis revealed that tritium occurs primarily in cytoplasm and rarely the nucleus of amebae. The most extensive cytoplasmic activity was observed during the initial 0–24 hr growth period of amebae as compared to later labeling periods. RNase or RNase followed by DNase extracted a large amount but not all label from amebae. These nucleases were least effective during the initial 24 hr period of growth. Thus it appears that tritium from uridine-5-H³ is not highly specific for RNA in amebae. However, the possibility that such label is associated with RNase-resistant RNA cannot be ruled out. More recent cytochemical studies do indicate the presence of RNase-resistant RNA in the cytoplasm of amebae. The activity found in penicillin-inhibited Bacteroides after uridine-5-H³ labeling and their reaction to the various digestive procedures was similar to amebae at corresponding labeling periods. Therefore at least some of the RNase-resistant material present in the cytoplasm of amebae may be derived from the ingested bacteria; this has been further found by appropriate experiments in which amebae were fed prelabeled bacteria. Nuclear activity when observed (always after 24 hrs growth) was associated either with the periphery of the nucleus and/or the endosome. It was not seen in the nuclear stroma. Some of this activity is RNase-resistant, perhaps representing double or multi-stranded RNA. It therefore appears that RNA is not distributed in the nuclear stroma in “long-term” labeling experiments.     

Entamoeba invadens: in vitro axenic encystation with a serum substitute

The current media for axenic cultivation of Entamoeba histolytica and Entamoeba invadens are supplemented with bovine or equine serum, which provides several essential nutrients to amoebas. Serum has also been considered an essential component in encystation media for E. invadens. A substitute of serum, PACSR has been described as an alternative for growth of E. histolytica and also maintains growth of E. invadens. When PACSR was used instead of serum for encystation of E. invadens the efficiency was the same as for serum. Our present data show that PACSR can support the growth and induction of encystation of E. invadens strain IP-1.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.     

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.     

Relationships between nucleic acid,nitrogen, and growth rate of tobacco cells in suspension culture

Summary Changes in the amount of nucleic acid and nitrogen, and the relationships between these amounts and the growth rate of tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2) at different initial nitrogen concentrations in the medium, were examined in batch cultures. During culture in basal medium, the amount of intracellular nucleic acid expressed per unit of dry biomass was 36.3 mg RNA g^–1 cell and 8.1 mg DNA g^–1 cell at the beginning of batch culture. These values increased 2.5 fold for RNA and 1.5 fold for DNA during the exponential growth phase and then gradually decreased with the decline in the growth rate. Similar changes were also observed in the medium containing less nitrogen. The specific growth rate, (day^–1), of the culture corresponded to the magnitude of the intracellular RNA content (mg RNA g^–1 cell), and the linear relationship, RNA=38+23 was obtained. In addition, there were remarkable positive correlations between the total and protein nitrogen, and during the cultures. The mononucleotide composition of total RNA (AMP+UMP)/(GMP+CMP) which was suggested to be a convenient index of metabolic activity was nearly constant (0.78 to 0.80) during tobacco cell culture in the basal medium.     

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.     

Circadian Rhythms of Nucleic Acid Metabolism in Neurospora crassa

Wild-type, band, and fluffy strains of Neurospora crassa exhibit circadian rhythms of ribonucleic acid and deoxyribonucleic acid content in the growth-front hyphae of cultures grown on a solid medium. There is also a rhythm of (3)H-uridine incorporation into the nucleic acids of the band strain. Maximum incorporation precedes the peaks of nucleic acid content which occur during conidiation. As cultures age, ribonucleic acid content decreases rapidly and deoxyribonucleic acid content decreases gradually in standing, shake, and bubble cultures. A reduction of ribonuclease activity with age is also noted in standing and shake cultures. The nucleic acid content, nuclease activity, and changes associated with age vary with the culture conditions.     

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.     

Secretory Hydrolases of Entamoeba histolytica1

ABSTRACT Cells of Entamoeba histolytica grown over a period of four days contained NADP⁺-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, β-N-acetylglucosaminidase, esterase, α-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP⁺-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as β-N-acetylglucosaminidase and α-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.     

Regulation of tropomyosin gene expression during myogenesis.

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.     

Entamoeba dispar: molecular characterization of the galactose/N-acetyl-d-galactosamine lectin

Amebiasis contributes to approximately 50 million cases of life-threatening dysentery worldwide. Comparison of the lectins from Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic) was undertaken to elucidate the differential roles of this molecule in invasion versus colonization. Surface lectin was less abundant on axenic E. dispar than on axenic E. histolytica, commensurate with differences in lectin (heavy and light subunits) RNA when assessed by semiquantitative RT-PCR. The 1G7 epitope, which falls within the immunodominant and immunoprotective cysteine-rich region (480-900), was absent on axenic E. dispar. Indirect immunofluorescence, transient transection of COS7, and immunoprecipitation demonstrated that the 1G7 epitope was conserved in the nonpathogenic lectin homologue but not exposed on live E. dispar trophozoites. Hgl2 (E. histolytica) and Dhgl2 (E. dispar) lectin homologues demonstrated comparable high-affinity binding to multivalent GalNAc(19) BSA. These data provide evidence for relative gene and conformational regulation of the E.dispar lectin.     

Pathogenic protozoans in man: differential characterization of Entamoeba histolytica/Entamoeba dispar complex,Acanthamoeba spp., Microsporidia

The review summarizes the results in the main parasitological topics of our Lab: amoebic infections due to Entamoeba histolytica/Entamoeba dispar complex and to Acanthamoeba spp. respectively, and human infections caused by microsporidia. Different rapid and advanced techniques have been included in the standardized diagnostic protocols for each topic, and a critical comparison among them was made, in order to define the gold standard diagnostic method: a) E. histolytica/E. dispar: in vitro culture, zymodeme typization, biomolecular identification (PCR), immunoenzymatic assay (ELISA) for direct detection in stools of specific surface antigenic lectins; b) Acanthamoeba spp.: in vitro culture, light and ultrastructural characterization, species identification by immunofluorescence method with monoclonal antibodies, in vitro pharmacological studies; c) Microsporidia: ultrastructural (TEM), biomolecular (PCR), biochemical and immunological (SDS-PAGE, Immunoblotting) studies for species identification, use of advanced ultrastructural techniques ("freeze-etching", "deep-etching") in order to deepen the spore wall structure, to study the cytoskeletal function of actin and to define the mode of infection, in vitro pharmacological assays on some inhibitors of chitin-synthases.     

Effect of jasplakinolide on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens

The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding.     

Axenic Mass Cultivation of Entamoeba invadens and Cell Membrane Isolation

SYNOPSIS. A method for axenic mass cultivation of Entamoeba invadens is described. A known monophasic culture medium for E. histolytica was modified by mixing it with a medium for Acanthamoeba castellanii and adding ferric chloride to provide more particulate material. This results in a high number of cells per ml after a relatively short culture period.
A cell membrane fraction of such a mass culture of E. invadens can be prepared by simple disruption of the cells and by low speed centrifugation. Owing to the lack of membrane-bounded organellae in the amebic cytoplasm there are only 2 kinds of membranes to be separated: the plasmalemma and the vacuole membranes.