Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Refractoriness of erythrocytes infected with Plasmodium falciparum gametocytes to lysis by sorbitol

Unlike erythrocytes infected with mature asexual parasites of Plasmodium falciparum, those infected with gametoeytes are not lysed by 5% sorbitol solutions. This observation was used to devise a method for producing synchronized cultures of gametocytes, free of asexual stage parasites. The refractoriness to sorbitol suggests that the major anion transport pathway, which appears in the membrane of erythrocytes infected with asexual stage parasites, is not present in cells infected with gametocytes.     

Free-flow electrophoresis for the separation of malaria infected and uninfected mouse erythrocytes and for the isolation of free parasites (Plasmodium vinckei): a new rapid technique for the liberation of malaria parasites from their host cells.

After aggregation of erythrocytes from malaria infected mice, the parasites (Plasmodium vinckei) could be set free using gentle mechanical forces. The mixture of freed parasites, infected and non-infected erythrocytes, and membraneous material was separated by free-flow electrophoresis. The free parasites produced were very pure and infectious. Morphological and enzymatic data on the separated fractions are presented. Free-flow electrophoresis also allowed the separation of infected and uninfected erythrocytes.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.     

Plasmodium berghei: Osmotic fragility of malaria parasites and mouse host erythrocytes

The osmotic properties of intraerythrocytic and ultrasonically liberated malaria parasites (Plasmodium berghei) were analyzed and compared with those of mouse host erythrocytes utilizing a multiple tube fragility test. Cells were incubated in phosphate buffered saline solutions of varying osmolalities ranging from 20–4000 mOsm. Changes in cell ultrastructure and parasite infectivity were used as indicators of osmotic damage. Intraerythrocytic and host cell-free plasmodia showed similar patterns of cell alteration and changes in infectivity following osmotic stress. The various developmental forms within each of the preparations responded somewhat differently to hypo-osmotic stress, however. The majority of merozoites seemed to be more sensitive than many trophozoites, schizonts, and segmenters. Small trophozoites were, on the average, more resistant than other developmental forms. Incubation of parasite populations in hypotonic salt solutions with osmolalities slightly greater than the infectivity threshold of 100 mOsm lysed the majority of the merozoites, whereas many small trophozoites were still intact. While normal erythrocytes were more resistant to hypo-osmotic stress than were either intracellular or free parasites, the majority of parasitized erythrocytes was less resistant than normal erythrocytes. The predominant alteration induced by hyperosmotic stress appears in the parasite's nuclear region with myelination of the nuclear membranes and chromatin clumping. The infectivity threshold in the hypertonic range was found to be approximately 2500 mOsm. Results indicate that these obligate intracellular parasites have a wide range of osmotic sensitivities and that they are capable of existing for short periods in various osmotic environments ranging from 100–2500 mOsm without complete loss of infectivity. This suggests that these parasites have osmotic regulatory capabilities at least comparable to those of host cells.     

Reevaluation, using marker enzymes, of the ability of saponin and ammonium chloride to free Plasmodium from infected erythrocytes

Saponin and ammonium chloride lysis have been applied for some time to the separation of erythrocyte membranes from malarial-infected erythrocytes, allowing easy isolation of the parasites. We present a reevaluation of the use of saponin and ammonium chloride as tools for isolating Plasmodium (knowlesi or falciparum) parasites. Acetylcholine esterase (EC 3.1.1.7) was used as an erythrocyte membrane marker and CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) as a parasite membrane marker to monitor fractionation by these agents. Both saponin and ammonium chloride produced hemolysis of uninfected and infected erythrocytes, but failed to separate host erythrocyte membrane from the parasite, regardless of its stage. Thus, saponin and ammonium chloride can be used to isolate whole infected erythrocytes, depleted of hemoglobin, by selective disruption of uninfected cells.     

Measurement of protein by dye-binding assay in the presence of metrizamide

Quantitative determination of protein using the binding of Coomassie Brilliant Blue G-250 was investigated with respect to interference with the density gradient material metrizamide, and compared with the corresponding interference using the Lowry method. The background absorption obtained with metrizamide in the absence of protein was less than 10% of that obtained with the Lowry method. In the presence of 0–4% metrizamide, parallel standard curves were obtained with 0–67 μg of protein in the samples. The curves overlapped in the range 0–40 μg of protein when metrizamide was included in the blanks. With up to 2% final concentration of metrizamide in the assay, the curves overlapped at all protein concentrations tested (0–67 μg). Correction for metrizamide interference is thus a simple procedure and a precise estimation of the metrizamide concentration is less critical than when the Lowry assay is used. The method is well suited for quantitation of protein in samples collected from metrizamide grandients.     

Isolation of <Emphasis Type="Italic">Plasmodium berghei</Emphasis> (Malaria) Parasites by Ammonium Chloride Lysis of Infected Erythrocytes

IMMUNOLOGICAL, biochemical and other studies of intra-erythrocytic parasites, such as malaria, would be greatly facilitated if the parasites could be obtained free from erythrocytes. To lyse infected erythrocytes, hypotonic shock¹, sonication², rapid pressure release obtained with the French press³, proteolytic enzyme digestion^4,5, saponin^6,7 and antibody-induced lysis^8,9 have been tried, but these either fail to remove erythrocyte debris completely, or cause structural damage to the organisms.     

Factors affecting the survival of frozen-thawed mouse spermatozoa

Mouse epididymal spermatozoa were frozen in solutions containing various compounds with different molecular weights, and the factors affecting the postthawing survival were examined. Monosaccharides (glucose, galactose) had almost no protective effect regardless of the concentration and the temperature of exposure. On the other hand, disaccharides (sucrose, trehalose) and trisaccharides (raffinose, melezitose) resulted in higher survival rates, especially at a concentration of around 0.35 mol/kg H(2)O (0.381-0.412 Osm/kg). Macromolecules, such as PVP10, Ficoll 70, bovine serum albumin, and skim milk had almost no effect, but compounds with a molecular weight of about 800, such as metrizamide and Nycodenz, had some protective effect. When a raffinose solution was supplemented with 10% metrizamide, resulting in an osmolality of approximately 0.400 Osm/kg, a high survival rate was obtained. Solutions at about 0.400 Osm/kg containing trehalose alone, trehalose + metrizamide, raffinose alone, and raffinose + metrizamide, were all effective for sperm freezing; frozen-thawed sperm could fertilize oocytes, and the resultant embryos could develop to live young after transfer. For freezing mouse spermatozoa, aqueous solutions at approximately 0.400 Osm/kg containing a disaccharide or a trisaccharide seem to be effective.     

The effect of purified aminoaldehydes produced by polyamine oxidation on the development in vitro of Plasmodium falciparum in normal and glucose-6-phosphate-dehydrogenase-deficient erythrocytes.

Purified aminoaldehydes produced by polyamine oxidation were toxic to the malarial parasite, Plasmodium falciparum, cultured in human erythrocytes. There was a profound effect on young ring forms, and, during maturation, parasites became more sensitive to the aldehydes. Oxidation of the aldehydes abolished the lethal effect. The plasmodia within glucose-6-phosphate-dehydrogenase (G6PD)-deficient erythrocytes were more sensitive to mono- and di-aldehydes than were parasites in normal erythrocytes. G6PD-deficient erythrocytes were also more sensitive to pretreatment with the dialdehyde produced by the oxidation of spermine. Pretreatment prevented further invasion by the parasites.     

Competitive Inhibition of Human Brain Hexokinase by Metrizamide and Related Compounds

Abstract: We have compared the competitive inhibitory effects of 2-deoxyglucose, glucosamine, N -acetylglucosamine, N -benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2-[3 - acetamido - 2,4,6 - triiodo -5-( N - methylacetamido) benzamido]-2-deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with K _is of 2.8 and 2.5 m M , respectively, for the mitochondrial and soluble forms. Glucosamine exhibited K _is of 0.58 and 0.29 m M , while 2-deoxyglucose exhibited K _is of 0.074 and 0.15 m M and N -acetylglucosamine 0.098 and 0.092 m M for these two forms, respectively. N -Benzoylglucosamine was by far the most effective inhibitor tested, with K _i values of 0.0086 and 0.022 m M , respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N -acetylglucosamine, 2-deoxyglucose, and N -benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2-deoxyglucose, N -acetylglucosamine, and N -benzoylglucosamine. Since N -benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N -benzoylglucosamine. However, gas chromatography-mass spectrometry analysis of metrizamide did not indicate the presence of N -benzoylglucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze-dried eluate fractions localized the inhibitory effect to the metrizamide peak.     

Metrizamide in D2O — A novel solute for the separation of labeled and unlabeled proteins by equilibrium density gradient centrifugation

Metrizamide(2-(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-d-glucose) dissolved in D₂O was found to be a very suitable medium for the separation of labeled and unlabeled proteins by equilibrium gradient sedimentation. It is nontoxic, and has little influence on the activity of enzymes. Solutions in the density range of 1.3–1.45 g cm^?3 have low viscosities. Since the spontaneous equilibrium gradient, which is dependent on the angular velocity, occurs only after a long time of centrifugation in metrizamide solutions, the equilibrium density gradient sedimentation of proteins can be performed at the highest available speed with any preformed shallow gradient. Examples for the separation of proteins of different densities are given.     

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.     

Separation and concentration of schizonts of Plasmodium falciparum by Percoll gradients

A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. II. Changes in Erythrocytes Infected with Babesia bigemina and B. argentina

SYNOPSIS. Cytoplasmic stippling, intensification of the cell margin, and alterations in color, which have been reported in erythrocytes parasitized by Plasmodium falciparum in man, have been seen also in bovine erythrocytes parasitized by either Babesia bigemina or B. argentina. These changes appear to be identical in the human and bovine infections.
Tests with each component of Giemsa stain in simple aqueous solutions alone and in various combinations with eosin, together with tests with Giemsa stains containing one azure component, showed that demonstration of the changes depends on the presence of azure A and eosin and on prolonged staining times at pH 7.2 to 7.4. Specific tests suggested that the changes represent catabolic by-products of the parasites.     

Metrizamide dissociates nuclear particles containing heterogeneous nuclear RNA

Metrizamide gradients were tested for the possible fractionation of the constitutive units of nuclear particles. Material from 35-55 S monoparticles was indeed distributed along the gradient but rerun experiments, CsCl density determinations, formaldehyde fixation prior to centrifugation suggested that the separation was due to dissociation or (and) action of endogeneous ribonucleases rather than to monoparticle fractionation. That dissociation had indeed occured was confirmed by the study of 60-110 S polyparticles. They were dissociated into ribonucleoproteins rich in phosphoproteins and into free proteins. These products were essentially similar to those obtained after NaCl treatment of the particles though the modes of action of metrizamide and NaCl are likely to be different. The loss of proteins from particles reaches 60-70% and we conclude that metrizamide gradients are not utilizable for the fractionation of the units of nuclear particles.     

Adenosine Triphosphate and the Pyruvic and Phosphoglyceric Kinases of the Malaria Parasite Plasmodium lophurae

SYNOPSIS. Adenosine triphosphate (ATP) with pyruvate, or adenosine diphosphate with phosphoenolpyruvate, favor the development of Plasmodium lophurae removed from its host erythrocytes and kept extracellularly in vitro. It seemed possible that the parasites might be deficient in enzymes of the glycolytic cycle concerned with the generation of ATP. The ATP content of duck erythrocytes infected with P. lophurae was lower than that of uninfected cells. Infected erythrocytes, however, had somewhat higher contents of both pyruvic kinase and phosphoglyceric kinase than did uninfected ones. Both of these enzymes could be found in the free parasites. Furthermore, the pyruvic kinase of the free parasites was inactivated by freezing and thawing, whereas that of the host erythrocyte was not affected. It will be necessary, therefore, to look further for the basis for the favorable effect of ATP with pyruvate on parasites developing extracellularly in vitro.     

Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.     

Concentration and Enzyme Content of In Vitro-Cultured Babesia bigemina-Infected Erythrocytes1

ABSTRACT. Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes-lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase-were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.