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PubMedDate not supplied

Zymodeme alteration of Entamoeba histolytica isolates under varying conditions.

Cysts of Entamoeba histolytica obtained from 2 asymptomatic subjects were cloned in vitro and the isoenzyme patterns and virulence of the cultures derived from them were determined. Incubation in Diamond's TYI-S-33 medium with Crithidia sp. for 80 d resulted in a change from zymodeme III to zymodeme II, with consistent virulence. Axenization by replacing Crithidia sp. with crithidial lysate had no further effect on the zymodeme but the virulence of the amoebae was attenuated. A reversal to zymodeme III, together with augmentation of virulence, was observed on the 20th day after transfer of axenic amoebae to Diamond's TYSGM-9 medium containing the original bacterial flora. The possibility that the isoenzyme pattern determining the zymodeme and the virulence behave as 2 independent variables in E. histolytica is discussed.

Animals↗
PubMedDate not supplied

A beta 1 integrin-like molecule in Entamoeba histolytica.

Human invasive amoebiasis is highly destructive, causing rapid necrosis and liquefaction of all tissues reached by the trophozoites. Degradation of extracellular matrix components (EMC) has been demonstrated during invasion of the basal lamina. Pursuing the idea that trophozoites might behave similarly to other invasive cells with respect to their interaction with EMC, plasma membrane proteins biochemically or functionally related to integrins were looked for. A 140 kDa molecular mass membrane protein from Entamoeba histolytica trophozoites with the characteristics of a beta 1 integrin-like fibronectin receptor was identified.

Animals↗
PubMedDate not supplied

Chemotherapy of Entamoeba histolytica: studies in vitro with bacitracin and its zinc salt.

Twenty-one Entamoeba histolytica isolates from a range of geographical locations and representing 3 different zymodemes were tested under xenic anaerobic conditions for their sensitivities to bacitracin and bactracin zinc. Isolates were over 13 times more sensitive to bacitracin zinc than to bacitracin. Our results indicate that bacitracin zinc may form the basis of an alternative non-toxic treatment of luminal amoebiasis.

Animals↗
PubMedDate not supplied

Transmission of Entamoeba histolytica within a family complex.

A limited outbreak of symptomatic intestinal and extraintestinal amoebiasis within a family complex is described. The infection was almost certainly transmitted by a Philippino housemaid, who was an asymptomatic carrier of Entamoeba histolytica infection acquired in her native country. Starch-gel electrophoresis showed isoenzyme patterns characteristic of pathogenic zymodeme XIX in all the amoebic isolates.

Adult↗
PubMed1981

Ribonuclease of Entamoeba invadens.

Two bands of RNAase activity were observed on gel electrophoresis of fractions obtained during partial purification of the RNAase of Entamoeba invadens. The faster migrating band which was responsible for most of the RNAase activity was isolated by gel electrophoresis. A pI of 5.5 was determined for this RNAase activity and estimates of its molecular weight and sedimentation coefficient gave values of about 25 000 and approximately 2.8 S. It exhibited maximum activity around pH 6, and digestion of RNA showed a pattern expected of an endoribonuclease.

Animals↗
PubMed1981

A calcium regulated adenosine triphosphatase in Entamoeba histolytica.

Axenically grown trophozoites of Entamoeba histolytica (NIH-200 strain) contain an active ATPase (170 nmol PO4/min per mg protein) with maximal activity at pH 8.8, a high affinity for ATP (Km approximately 40 micro M) and an absolute and specific requirement for Ca2+. The activation by Ca2+ shows positive cooperativity (nH = 2.48) at calcium concentrations below 8 micro M and no cooperativity between 8 and 25 micro M. The latter concentration fully saturates the enzyme. The observed activity is insensitive to oligomycin, ouabain and ruthenium red and is unaffected by a range of inhibitors of electron transport and uncouplers of oxidative phosphorylation. The enzyme exhibits structure bound latency and is tightly bound to cellular membranes. It is sedimentable ( greater than 80%) by high speed centrifugation of cell homogenates which are either protected osmotically or in which subcellular structures are damaged by sonication or treatment with Triton X-100. Arrhenius plots of V in the temperature range of 0-38 degrees C are linear without breaks, similar to other pyrophosphatases of E. histolytica. The calculated activation energy is 14.8 kcal/mol. This finding as well as the failure of phospholipase treatment to affect activity indicate that interactions with membrane lipids play no role in the catalytic function of this tightly membrane-bound ATPase.

Animals↗
PubMed1981

Susceptibility of Entamoeba histolytica to oxygen intermediates.

To explore the susceptibility of the extracellular protozoan, Entamoeba histolytica, to toxic oxygen intermediates, trophozoites were exposed to fluxes of O2, H2O2, and OH. generated enzymatically by the glucose oxidase and xanthine oxidase reactions. HM-1 trophozoites were resistant to O2, but were readily killed by H2O2 alone. OH. and 1O2 were not required for effective amebicidal activity. The addition of peroxidase and halide enhanced trophozoite killing by H2O2. Sonicates of amebae contained virtually no catalase and little glutathione peroxidase activity which may contribute to susceptibility to H2O2. Coupled with our previous studies with Toxoplasma gondii and Leishmania spp. these observations indicate that there is a broad spectrum of susceptibility of intra- and extracellular pathogenic protozoa to killing by oxygen intermediates.

Animals↗
PubMed1983

Preliminary characterization of ribosomes of Entamoeba invadens.

The ribosomes of Entamoeba invadens trophozoites have sedimentation coefficients of 77, 53 and 36 S. Most of the ribosomal proteins are basic and their one- and two-dimensional electrophoretic patterns differ from the corresponding patterns of Escherichia coli and Saccharomyces cerevisiae. Two dozen bands were observed in the 10 000 to 100 000 molecular weight range following sodium dodecylsulfate-gel electrophoresis of amoebal ribosomal proteins. Long, thin pronase-sensitive structures were seen in electron micrographs of E. invadens ribosomal preparations.

Animals↗
PubMed1983

Degradation of biogenic oligosaccharides by beta-N-acetyl-glucosaminidase secreted by Entamoeba histolytica.

beta-N-Acetylglucosaminidase secreted by Entamoeba histolytica was extracted from the growth medium by affinity chromatography on CH-Sepharose 4 B coupled to p-aminophenyl-1-thio-beta-2-acetamido-2-deoxyglucopyranoside. The enzyme was further purified by isoelectric focusing, by sequential chromatography on DEAE-cellulose and Sephadex G-150, and by preparative disc gel electrophoresis. Chitobiose (betaGlcNAc1-4GlcNAc) derived from chitin as well as the oligosaccharides betaGlcNAc1-4 betaGlcUA1-3GlcNAc, betaGlcNAc1-4 betaGlcUA1-3 betaGlcAc1-4GlcUA, and betaGlcNAc1-4 betaGlc-UA1-3 betaGlcNAc1-4 betaGlcUA1-3 betaGlcNAc1-4GlcUA derived from hyaluronic acid were tested as potential physiological substrates. All these oligosaccharides are susceptible to action of beta-N-acetylglucosaminidase from E. histolytica. Under identical conditions chitobiose is cleaved 38-48 times faster than hyhyauronate oligosaccharides. No release of N-acetylglucosamine was observed when glycopeptides from ovalbumin were used as substrate. The pH optimum of hydrolase activity was 4.5 when chitobiose was used as substrate. Optimal hydrolysis of aluronate oligosaccharides was observed at pH 3.0 for trisaccharide and pH 2.0 for tetra- and hexasaccharide, respectively. Estimation of molecular weight by means of gel filtration gave values of 75 000. The isoelectric point was 5.02 beta-N-Acetylglucosaminidase from E. histolytica does not act on macromolecular chitin and hyaluronic acid.

Acetylglucosaminidase↗
PubMed1983

Separation and characterization of two UTP-utilizing hexose phosphate uridylyltransferases from Entamoeba histolytica.

Two UTP-utilizing uridylyltransferases which react with both glucose 1-phosphate and galactose 1-phosphate were isolated from cell-free extracts of Entamoeba histolytica. The more specific of these enzymes, glucose-1-phosphate uridylyltransferase, acts preferentially on glucose 1-phosphate, having a maximum velocity 20-fold greater with this substrate than with galactose 1-phosphate. It was purified 200 fold with a 25% yield and has a molecular weight of 45 000. This enzyme requires a reducing agent for stability. The less specific transferase reacts with both hexose phosphates, having a maximum velocity of 1.35 times greater with galactose 1-phosphate. It was purified 1000 fold with a 20% yield, and has a molecular weight of 40 000. The common Leloir enzyme, UDP glucose-hexose-1-phosphate uridylytransferase (EC 2.7.7.12), was not found in this organism. To avoid confusion with the Leloir enzyme our experience suggests that the less specific enzyme, which is presently referred to in the literature as galactose-1-phosphate uridylyltransferase (EC 2.7.7.10), should be named UTP:hexose-1-phosphate uridylyltransferase (EC 2.7.7.?). The more specific enzyme (EC 2.7.7.9) should be more clearly named UTP:glucose-1-phosphate uridylyltransferase.

Entamoeba histolytica↗
PubMed1984

The occurrence of beta-amylase in Entamoeba histolytica.

A beta-amylase like activity isolated from a cell homogenate of Entamoeba histolytica, and separated from other glucohydrolytic activities by gel filtration and isoelectric focusing, hydrolyzed amylose, amylopectin and glycogen, yielding maltose as reaction product. From the non-reducing ends of the 4-nitrophenyl-alpha-D-glycosides of maltotriose, maltotetraose, maltopentaose and maltohexaose the enzyme removed maltose units. The pH optimum for hydrolyzing the poly- and oligosaccharides was pH 6.5, and the temperature optimum was 29 degrees C. Estimation of relative molecular mass by gel filtration on Sephadex G 75 gave values around Mr = 32 000. The isoelectric point was found to be 5.7.

Amylases↗
PubMed1984

Isolation and characterization of RNA of Entamoeba histolytica.

RNAs were isolated from Entamoeba histolytica with a high salt sodium dodecylsulfate-diethylpyrocarbonate technique. Majority species of 25 S, 17 S and 4 S RNAs were detected after sucrose gradient centrifugation. An additional 5 S RNA was detected by polyacrylamide gel electrophoresis. The molecular weights of these RNAs as determined by completely denaturing polyacrylamide gel electrophoresis were 1.31 X 10(6) (25 S), 0.803 X 10(6) (17 S), 4.0 X 10(4) (5 S) and 2.5 X 10(4) (4 S). The 25 S RNA was labile and dissociated under mild denaturing conditions (between 37 degrees C and 55 degrees C) into 17 S and 16 S RNAs with molecular weights of 0.700 X 10(6) and 0.614 X 10(6), respectively; under completely denaturing conditions an additional 5.8 S RNA with a molecular weight of 4.8 X 10(4) was detected. Evidence is presented which suggests that the lability of the 25 S RNA is the result of an in vivo cleavage rather than one which is generated during RNA isolation.

Animals↗
PubMed1985

Isolation and characterization of Entamoeba histolytica mutants resistant to emetine.

Isolation of emetine-resistant mutants of Entamoeba histolytica is described. Spontaneous rate of mutation, obtained from a Luria-Delbrück fluctuation test was 2.5 X 10(-7). The mutagenesis frequency obtained from the number of colonies grown in the presence of emetine, divided by the number of viable trophozoites inoculated in semisolid agar was less than or equal to 10(-7) and it was increased from 10 to 100 fold with ethyl methanesulfonate. Two types of clones were isolated: one was cross-resistant to colchicine and the other was not, indicating that mutation took place at different loci. Protein synthesis in the presence of emetine remained unaltered for colchicine-sensitive mutants, suggesting that the molecular lesion lay in the protein synthesis machinery. Colchicine-resistant mutants showed a lower level of [35S]methionine incorporation, probably due to alterations in the molecular transport of some substances across the membrane. This mutant clone was also deficient in red blood cell adhesion, suggesting membrane alterations.

Animals↗
PubMed1985

Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens.

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.

Animals↗
PubMed1985

Subcellular fractionation of amoebapore and plasma membrane components of Entamoeba histolytica using self-generating Percoll gradients.

Separation and initial characterization of subcellular organelles from Entamoeba histolytica have been achieved by the use of self-generating Percoll gradients. Adequate resolution was obtained within one hour of amoeba homogenization. The ion-channel forming activity, amoebapore, was found associated with highly dense, small diameter particles, that were resolved from the numerous digestive vacuoles, and the plasma membrane. Following iodination of intact trophozoites with lactoperoxidase, the label was found to be incorporated into two distinct sedimentable fractions. The major component contained the near totality of the concanavalin A binding glycoproteins and the 5-iodonaphthalene-1-azide labelled intrinsic membrane proteins. It therefore was identified as the plasma membrane. The minor lactoperoxidase-iodinated component was found in the upper section of the gradient, associated with a particulate fraction nearly devoid of concanavalin A-binding glycoproteins. This fraction appears to represent particulate material on the external surface of the amoeba that is distinct from the plasma membrane. The digestive vesicles, which were identified by the presence of acid phosphatase and beta-D-glucosaminidase activities, appeared in the gradient as one main peak with a shoulder in the region of the plasma membrane. Analysis of the polypeptides and their labelling pattern in each fraction of the gradient are presented. Distinctive characteristics are discussed including the identification of a unique soluble protein highly labelled by 5-iodonaphthalene-1-azide.

Animals↗
PubMed1986

Studies on the substrate specificity of alpha- and beta-amylase of Entamoeba histolytica.

From the supernatant fraction of cell homogenates of Entamoeba histolytica alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and beta-amylase (1,4-alpha-D-glucan maltohydrolase, EC 3.2.1.2) were separated and purified by gel filtration and isoelectric focusing, followed by DEAE- and CM-chromatography, respectively. Both enzymes catalyzed the degradation of amylose, amylopectin and glycogen. Hydrolysis of polysaccharides by alpha-amylase yielded as reaction products maltose, maltotriose, maltopentaose and maltohexaose, but no free glucose. beta-Amylase produced as main degradation product of glucopolysaccharides maltose and to a minor extend maltotriose, but no glucose. alpha- and beta-amylase were able to hydrolyze 4-nitrophenyl-alpha-D-glucooligosaccharides (Gn-pNP) containing more than two glucose units per molecule. Under identical conditions G6-pNP was cleaved at highest velocity by alpha-amylase, while beta-amylase exhibited the highest activity with G4-pNP as substrate. alpha-Amylase hydrolyzed G4-pNP, G5-pNP and G6-pNP yielding as main reaction product G2-pNP, but also the formation of G1-pNP and G3-pNP from G4-pNP, of G1-pNP, G3-pNP and G4-pNP from G5-pNP, of G1-pNP, G3-pNP, G4-pNP and G5-pNP from G6-pNP was observed. alpha-Amylase as endo-glucohydrolase attacked all glycosidic bonds in G6-pNP, G5-pNP and G4-pNP, while beta-amylase successively removed maltose units from the non-reducing ends of the glycosides.

Amylases↗
PubMed1986

Cysteine proteinase of Entamoeba histolytica. I. Partial purification and action on different enzymes.

A method for a 50-60-fold purification of a cysteine proteinase from trophozoites of Entamoeba histolytica using 35-80% ammonium sulphate fractionation, gel chromatography on Sephadex G-75, and preparative isoelectric focusing is described. The enzyme was examined for its proteolytic potencies towards native enzyme substrates. The amebic proteinase directly inactivates aldolase and glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle as well as glucose-6-phosphate dehydrogenase from yeast. The inactivation of citrate synthase from porcine heart proceeds rather slowly, whereas malate dehydrogenase from porcine heart is not affected by the amebic proteinase under the condition used. With the exception of aldolase all inactivated enzyme substrates have been cleaved by limited proteolyses yielding major cleavage products. The inactivation of aldolase probably functions by the release of a small segment from a terminus being essential for aldolase activity.

Animals↗
PubMed1986

Cysteine proteinase of Entamoeba histolytica. II. Identification of the major split position in bovine insulin B-chain.

The substrate specificity of a cysteine proteinase of Entamoeba histolytica was tested using the oxidized B-chain of bovine insulin. 28 proteolytic peptides were formed after different intervals of incubation and could be separated by high-performance liquid chromatography. Two dominant peptides appearing after 10 min of incubation were isolated and analyzed for amino acid composition. The results obtained indicate that these two peptides were formed by cleavage of the peptide bond between the amino acids 23 and 24. Thus as major split position the Gly-Phe bond in the insulin B-chain could be identified.

Amino Acids↗