Enrichment and biochemical characterization of Toxoplasma gondii tachyzoite plasmalemma.

The protozoan parasite Toxoplasma gondii possesses a triple surface membrane called the pellicle. This is made of an outer plasmalemma and an inner membrane complex lying under the plasmalemma. Using a high salt glycerol treatment followed by sonication, we have obtained a partial dissociation of the pellicle. A plasmalemma-enriched fraction was isolated on 0.7 M sucrose. It was identified by immunodetection of the tachyzoite major surface antigens. Protein content, resolved by SDS-PAGE, revealed that the surface protein SAG1 is the major component of the plasmalemma. The plasmalemma fraction is made of small vesicles (20-100 nm) which possess a low density (1.085-1.090 g/cm3 in sucrose) contrasting with other eukaryotic plasma membranes (1.12-1.16 g/cm3).

[The membrane complex of Toxoplasma gondii: ultrastructural study of its constituents]. / Le complexe membranaire de Toxoplasma gondii: Etude ultrastructurale de ses constituants.

Toxoplasma gondii membrane complex was fragmented by sonication and separated on a sucrose continuous gradient. Ultrastructural analysis of the 1,08 g/cm3 density fraction (that which corresponded with a maximal detection by the monoclonal antibody plasmalemma marker) showed an homogeneous population of small unimembrane vesicles. Ultrastructural analysis of the 1,13 g/cm3 density fraction (that with corresponded with a maximal detection by the monoclonal antibody inner complex marker) showed an heterogeneous population with unimembrane vesicles and trimembrane fragments.

Isolation of the pellicle of Toxoplasma gondii (Protozoa, Coccidia): characterization by electron microscopy and protein composition.

Two methods were compared for the isolation of the Toxoplasma gondii pellicle: centrifugation using a discontinuous sucrose gradient and affinity purification by microspheres activated with protein A. Electron microscopy showed that these two means of purification gave similar results, although better yields were obtained with the former than with the latter. Proteins of membrane fractions isolated using sucrose gradient were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands in the molecular-weight regions of 19, 22, 35, and 79 kDa appeared to be associated with the T. gondii pellicle.

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study.

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.

Toxoplasma gondii: demonstration of intrinsic peroxidase activity during lacto-peroxidase mediated radioiodination of tachyzoites.

Tachyzoites of Toxoplasma gondii have been radioiodinated under various conditions with or without lactoperoxidase, with glucose oxidase being used to generate hydrogen peroxide. Erythrocytes were iodinated simultaneously as a control. In our conditions, tachyzoites were more intensely labelled in the absence of lactoperoxidase. This result can be explained by the existence of an intrinsic peroxidase activity which interfere with the exogenously added enzyme during surface radioiodination.

Purification of Toxoplasma gondii suspensions from mice peritoneal exudate.

Purification procedures of Toxoplasma gondii suspensions are described. These methods were designed for the elimination of leucocytes or TG 180 cells. We have been using either gradient centrifugation in Ficoll or gel filtration. In the first case, only leucocytes are eliminated (95% of the initial content). On the other hand, gel filtration allowed the elimination of TG 180 cells. The yield (67% to 98%) depends on the chosen gel (Ultrogel AcA 202 or Sephadex G25 fine), and on the amount of TG 180 cells present before filtration.

[Study on resistant forms of Trichomonas vaginalis (author's transl)]. / Contribution à l'étude des formes de résistance de Trichomonas vaginalis.

We have demonstrated resistant forms of Trichomonas vaginalis studied their morphology and induced transformation into vegetative forms. This work has led to an hypothesis concerning the parasite cycle which may throw light on the problems of transmission and resistance to therapeutic agents.

[Isolation of "Toxoplasma gondii" membrane antigens using latex microspheres (author's transl)]. / Isolement de fractions antigéniques membranaires de Toxoplasma gondii par l'intermédiaire de microsphères de méthacrylate.

The authors describe a method for isolation of Toxoplasma gondii antigenic membrane components. Technical work is described into four points: 1) specific labelling of membrane proteins was realised by DD125ISA; 2) methacrylate microspheres were bound to unbroken toxoplasma membranes by the mean of an indirect technic with antibody molecules (double sandwich); these microspheres were used to modify the membrane density; 3) toxoplasmas bound to microspheres were broken by sonication; 4) microspheres fixed to membrane components were isolated by isopycnic ultracentrifugation on continuous sucrose gradient.

[Diagnosis of toxoplasmosis by indirect hemagglutination. I. Comparative study of fixation of glutaraldehyde on formolized erythrocytes of a soluble endogenous antigen, and insoluble particulate antigen and a total antigen]. / Diagnostic de la toxoplasmose par héhagglutination indirecte. I. Etude comparative de la fixation par le glutaraldéhyde sur des globules rouges formolés d'un antigène soluble, d'un antigène particulaire insoluble et d'un antigène total

Three different sorts of antigen are prepared ultrasonically: a soluble endogenous antigen, an insoluble particular antigen and a total mixed antigen. Formolinized red blood cells are sensibilized with glutaraldehyde. The authors study the sensitivity of the suspensions as a function of the different modes of antigen preparation. The results are compared with those obtained by immunofluorescence. The authors show the usefulness of total ultrasonic antigen which provides suspensions of great sensitivity easily and economically prepared.

[Diagnosis of toxoplasmosis by indirect hemagglutination. II. Value of mixed total antigen fixation in the presence of glutaraldehyde in the early diagnosis of the disease]. / Diagnostic de la toxoplasmose par hemagglutination indirecte. II. Intérêt de la fixation de l'antigène total mixte en présence de glutaraldéhyde dans le diagnostic précoce de l'affection

2640 sera were titered simultaneously with IHA and IFAT. The results obtained show a good correlation between the two technics used. The IHA test seems particularly useful in IgM detection and, according to the mode of operation, is able to detect lower titers of anti-toxoplasma antibodies than in the case of IFAT.