Trypanosoma cruzi: Surface charge and freeze-fracture of amastigotes

EXPERIMENTAL

PARASITOLOGY

Trypanosoma

TBCIA ULISSESDE

59, 12-23 (1985)

cruzi:

CARVALHO,

Surface Charge of Amastigotes

and Freeze-Fracture

THAIS SOUTO-PADR~ON,ANDWANDERLEYDESOUZA'

Institute de Biofisica, Universidade Federal do Rio de Janeiro, Ilha do Funddo, Rio de Janeiro, RJ 21941, Brazil (Accepted for publication 12 June 1984) DE CARVALHO, T. U., SOUTO-PADR~N, T., AND DE SOUZA, W. 1985. Typarzosoma cruzi: Surface charge and freeze-fracture of amastigotes. Experimental Parasitology 59, 12-23. Amastigotes of Tlypanosomcr cwzi, within vertebrate cells or isolated from the supernatant of vertebrate cell cultures (L-A9 fibroblast or J774G8 macrophage-like cell lines), possess glycoproteins or glycolipids on the cell surface according to the periodic acid-thiosemicarbazide-silver proteinate technique used in association with electron microscopy. The cell surface of isolated amastigotes is negatively charged, as evaluated by the binding of cationic particles (colloidal iron hydroxyde at pH 1.8 and cationized ferritin at pH 7.2) as well as by direct measurement of cellular electrophoretic mobility. Amastigotes (Y strain) isolated from the spleen of infected mice and amastigotes (Y and CL strains) from the supernatant of cell cultures previously infected with T. cruzi have the same mean electrophoretic mobility (- 0.85 pm see-’ V-’ cm). It is intermediate between the epimastigote and the trypomastigote forms (determined previously). Sialic acid is the important component responsible for the negative surface charge, as determined by the use of neuraminidase. Thus, it is possible to use the mean electrophoretic mobility as an indicator for identifying amastigotes of T. cruzi. D 1985 Academic Press, Inc. INDEX DESCRIPTORS: Trypanosoma cruzi; Protozoa, parasitic; Hemoflagellate; Amastigote, surface; Electron microscopy; Cytochemistry; Glycoprotein; Sialic acid; Surface charge; Electrophoretic mobility.

In the present investigation, we used ultrastructural cytochemistry, freeze-fracture, and determination of cellular electrophoretie mobility to characterize the cell surface of T. cruzi amastigotes isolated primarily from cultures of the J774G8 macrophage-like cell line (De Carvalho and De Souza 1983).

The cell surface of the epimastigote and trypomastigote forms of Trypanosoma cruzi has been analyzed in recent years (Colli et al. 1981; De Souza 1984). These studies became possible because of methods for obtaining large numbers of T. crui epimastigotes and trypomastigotes. For the amastigotes, there is little data on surface properties due to difficulties in obtaining parasites that can be identified as amastigotes with certainty. In the last years, however, methods have been developed to make it possible to carry out studies on this form (Abrahamson et al. 1983; Murray et al. 1982; Villalta and Kierszenbum 1982).

MATERIALS

The Y and CL strains of Trypanosoma cvuzi were used. Bloodstream trypomastigotes were maintained in Swiss albino mice by intraperitoneal inoculation. Trypomastigotes were collected on the 7th or 14th day of infection, which corresponds to the peak of parasitemia of the Y and CL strains, respectively. Blood was collected in test tubes with 3.8% sodium citrate, centrifuged at 1.5Ogfor 10 min, and left at 37 C for 30 min. The new pellet was discarded and the supernatant, containing the parasites, was collected and cen-

’ To whom correspondence should be addressed.

12 0014-4894185$3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

Trypanosoma

cruzi: AMASTIGOTE

trifuged at 95Ogfor 10 min. The pellet was then washed in Tyrode’s solution (composition, in g/liter: NaCI, 8.0; KCl, 0.2; MgCl,. 6H,O, 0.1; NaHCO,, 1.0; NaH,PO, . H,O, 0.05; CaCl,, 0.2; and glucose, 1.0). To isolate amastigotes, we used the cell lines L-A9 (transformed fibroblast) and J774G8 especially (a macrophage-like cell of tumor origin), as described previously (De Carvalho and De Souza 1983). Briefly, the cells were infected with bloodstream trypomastigotes, and the cultures were examined every day using an inverted microscope. When a large number of amastigotes were seen in the supernatant, they were isolated by centrifugation in a metrizamide gradient. Using this procedure, amastigotes from the Y and CL strains of T. cvuzi were obtained. Amastigotes were also isolated from the spleen of mice infected with the Y strain of T. cruzi as described previously (Carvalho et al. 1981). For electron microscopy, the infected cells (J744G8), as well as the amastigotes, were collected by centrifugation, and the pellet was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 30 min at room temperature. After fixation, the pellet was rinsed twice with buffer and postfixed for 1 hr at room temperature in 1% 0~0, in 0.1 M cacodylate buffer. After this postfixation, the pellet was cut into small pieces, dehydrated in ethanol, and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate, and were observed in a Jeol 100 CX electron microscope. For detection of carbohydrates; thin sections of 577468 cells, infected with T. cruzi or isolated amastigotes, were collected on gold grids and incubated for 20 min at room temperature in the presence of 1% periodic acid. After incubation, the grids were rinsed three times with distilled water, incubated in the presence of 1% thiosemicarbazide in 10% acetic acid for 30 min, and rinsed once in 10% acetic acid and twice in distilled water before incubation for 30 min in the dark in the presence of 1% silver proteinate. After incubation, the grids were rinsed several times in distilled water and were observed unstained with the electron microscope (Thiery 1967). For the localization of anionic sites on the surface of amastigotes of T. cruzi, only those obtained from the supernatant of the J774G8 cell line were used. The following techniques were employed: (1) Colloidal iron hydroxyde: after fixation in glutaraldehyde, the cell suspension was incubated for 30 min at room temperature in a dialyzed suspension of colloidal iron hydroxyde at pH 1.8 (Gasic et al. 1968); they were washed twice in 12% acetic acid and once in phosphate buffer prior to postfixation with 0~0,; and (2) cationized ferritin: after glutaraldehyde fixation, the cells were incubated for 30 min at room temperature in a suspension of cationized ferritin in 0.1 M phosphate buffer, pH 7.2; they were then washed twice in buffer,

SURFACE ANALYSIS

13

postfixed in OsO,, and processed as described above (Danon et al. 1972). For freeze-fracture, after glutaraldehyde fixation, the cells were washed twice in 0.1 M cacodylate buffer, pH 7.2, and then exposed to ascending concentrations of glycerol in cacodylate buffer until, after 30 min, a final concentration of 30% glycerol was attained. The cells remained in 30% glycerol for 3-12 hr. Specimens were mounted on Balzers support disks and were rapidly frozen in the liquid phase of Freon 22 cooled by liquid nitrogen. Freeze-fracture was carried out at - 115 C in a Balzers apparatus, and the specimens were shadowed with platinum/carbon at 2 . 10m6Torr immediately after fracturing. Replicas were cleaned with sulfuric acid and clorox, and were examined in a Jeol 100 CX electron microscope. Particle counts were made in electron micrographs enlarged to 100,000diameters. The electrophoretic mobility of the amastigotes was determined in a Zeiss cytopherometer with a current of 4-6 mA and a final voltage of 100 V. The cell suspension was placed into the chamber and then allowed to equilibrate for 10 min. Measurements were made at a temperature of 25 C in a 0.85% NaCl solution (ionic strength, 0.145 mol dme3, pH 7.2). When the current was switched on, we measured with a stop watch the time necessary for one cell to travel across two vertical lines, which corresponded to a distance of 16 pm. Then, the polarization was reversed and the same cell was measured again, travelling in the opposite direction. Fifty to one hundred cells were measured for each sample analyzed. In some experiments, the pH of the solution was varied from 2.0 to 11.O.The change in the pH was obtained by adding various amounts of 0.1 M HCl or NaOH to the NaCl solution. Calibration of the equipment was made by measuring the electrophoretic mobility of fresh human erythrocytes (0, Rh-). Amastigotes were washed twice in Tyrode’s solution and were then incubated for 30 min at 37 C in the presence of 0.2 U/ml neuraminidase (Sigma type X, from Clostridium perfiingens) in Tyrode’s solution, pH 6.0, or for 15 min at 37 C in the presence of 500 pgiml trypsin (Sigma, type III) in Tyrode’s solution, pH 7.2. The effect of trypsin was stopped by the addition of fetal bovine serum (10% final concentration) to the test tube. After enzyme treatment, the cells were collected by centrifugation, washed twice in Tyrode’s solution and fixed in 2.5% glutaraldehyde as described above. Enzyme-treated cells were used for cytochemical studies (incubation in the presence of colloidal iron hydroxyde and cationized ferritin particles) and determination of the cellular electrophoretic mobility. RESULTS

The general aspect of the macrophage-

14

DE CARVALHO,

SOUTO-PADRdN,

AND DE SOUZA

FIG. 1. General aspect of the macrophage-like cell line infected with Trypanosolna cruzi. Intracellular amastigotes (*) are seen. HCN, host cell nucleus. x 5800. FIG. 2. General aspect of the population of Trypanosoma cv~zi amastigotes isolated from the supernatant of cultured macrophage-like cells previously infected with bloodstream forms of the parasite. x 4500.

like cell containing intracellular forms of Trypanosoma cvuzi is shown in Fig. 1. Figure 2 shows the general aspect of the purified amastigotes as seen by electron microscopy. Examination of the structural organization of the isolated parasites indicated that they were similar to the amastigotes observed within the cells (not shown). The kinetoplast was rod-shaped and localized anterior to the nucleus. The parasites had a short flagellum, about 1 pm long, which protruded slightly from the flagellar pocket. Amastigotes in division could be observed. In all preparations examined, a small number of forms (apparently in transition to trypomastigotes) could be observed. These cells had a longer flagellum; the kinetoplast was located laterally in relation to the nucleus and showed a rod shape. The plasma membrane of the amastigotes was intact. In thin sections of cells routinely processed for transmission electron

microscopy, no clear surface coat was observed. However, a layer of reaction product was clearly seen on the surface of isolated (Fig. 4) or intracellular (Fig. 3) amastigotes when thin sections were submitted to the periodic acid-thiosemicarbazide-silver proteinate technique, which detects the presence of glycoproteins and/or glycolipids (Thiery 1967). The reaction was specific since no reaction product was observed on the surface of cells in the control sections, which were incubated in the presence of thiosemicarbazide and silver proteinate without previous oxidation by periodic acid (Fig. 5). Colloidal iron hydroxyde particles at pH 1.8 (Figs. 6, 7) and cationized ferritin particles at pH 7.2 (Fig. 8) became bound to the surface of T. cruzi amastigotes. The particles were seen distributed homogeneously throughout the cell surface, but did not penetrate the flagellar pocket. Thus, no cationic particles were seen associated with

Trypanosoma

cruzi: AMASTIGOTE

SURFACE ANALYSIS

FIGS. 3-5. Thin sections submitted to the periodic acid-thiosemicarbazide-silver proteinate technique for detection of carbohydrates. Reaction product is seen on the whole surface of T~ypanmoma cmzi amastigotes Located within the host cell (Fig. 3) or after purification (Fig. 4). No reaction product is seen on the surface of parasites from sections which were not oxidized by periodic acid (Fig. 5). Arrows indicate the parasite’s surface. F, flagellum. Figs. 3 and 5, X 17JOO; Fig. 4, x 21,000.

16

DE CARVALHO,

SOUTO-PADRON,

AND

DE SOUZA

Trypanosoma

cruzi: AMASTIGOTE

SURFACE ANALYSIS

17

the flagellar membrane or with the mem- tion in a metrizamide gradient from the supernatant of cell cultures. With the Y brane which lines the flagellar pocket (Fig. 7). With both labels for anionic sites, varstrain, we also measured the electrophoious layers of particles were seen on the cell retie mobility of amastigotes isolated from the spleen of infected mice. In all cases, the surface. Treatment of the amastigotes with neur- same mean electrophoretic mobility was aminidase inhibited the binding of colloidal obtained, showing a value of about -0.85 iron hydroxyde particles to the parasite sur- pm set -’ V-’ cm. (Table I). Figure 14 face (Fig. 11). The same treatment signifi- shows a population analysis of amastigotes cantly reduced the binding of cationized obtained from three different sources. It ferritin particles. Some cells showed a can be seen that the population was hetersingle layer of particles on the cell surface, ogeneous in terms of the electrophoretic while other cells showed no particles at all mobility of the cells. Values of electrophoretie mobility varying between -0.4 and (Figs. 9, 10). Treatment of the amastigotes with trypsin - 1.6 km set-’ V-t cm were observed significantly reduced, but did not com- mainly with the amastigotes isolated from pletely abolish, the binding of cationized the spleen of infected mice. The population ferritin and colloidal iron hydroxyde parti- most homogeneous was that obtained from cles to the cell surface of the parasite. the supernatant of the J774G8 macrophageAt low magnification, it could be seen by like cell line. All parasites showed a freeze-fracture that both protoplasmic and random movement toward the positive extracellular faces of the plasma membrane electrode; some moved with the anterior of T. cruzi had a low density of intramem- end toward the cathode, others with the branous particles (303 & 94 and 541 ? 33 posterior end, and others laterally. Figure 15 shows the effect of the pH of particles/km2 for the protoplasmic and extracellular faces, respectively) which were the solution in which the cells were suspended on the mean electrophoretic mohomogenously distributed. Few particles were seen on either face of the membrane bility of amastigotes of T. cruzi. At pH 2.0, of the short flagellum (Fig. 12). The cyto- amastigotes had a positive surface charge stome could be easily recognized as a re- migrating toward the negative electrode. At gion of the membrane delimited by a palli- pH values higher than 2.2, the cells had a sade of adjacent intramembranous particles negative surface charge which increased localized close to the flagellar pocket. A with the pH increase of the solution. Belarge number of particles was seen in the tween pH 7.0 and 10.0, the electrophoretic membrane lining the cytostome (Figs. 12, mobility did not vary significantly; it increased again at pH values above pH 10.0. 13). Based on the curve shown in Fig. 15, we Our observations on the electrophoretic mobility of T. cruzi amastigotes indicated determined that the isoelectric point for the that they had a net negative surface charge surface of T. cruzi amastigotes was 2.0 for migrating to the positive electrode. We an- cells suspended in a saline solution with a alyzed amastigotes from the Y and CL ionic strength of 0.145 mol dme3 at 25 C. strains which were purified by centrifugaTreatment of T. cruzi amastigotes with eiFIGS. 6-8. Trypanosoma cruzi amastigotes incubated in the presence of either colloidal iron hydroxyde particles at pH 1.8 (Figs. 6, 7) or cationized ferritin particles at pH 7.2 (Fig. 8) after glutaraldehyde fixation. Particles are seen on the whole surface of the parasite, but not in the portion of the cell body and flagellar membranes lining the flagellar pocket region (arrow in Fig. 7). Figs. 6 and 8, ~39,000; Fig. 7, x20,000.

18

DE

CARVALHO,

SOUTO-PADRdN,

AND

DE

SOUZA

Trypanosoma

cruzi: AMASTIGOTE

SURFACE ANALYSIS

19

FIGS. 12-13. Freeze-fracture replicas of isolated trypanosoma cruzi amastigotes. A low density of intramembranous particles is seen on the protoplasmic face of the membrane which lines the cell body and the short flagellum (F). The cytostome (*) clearly appears as a region delimited by a linear array of particles (arrows). Fig. 12, x 35,000; Fig. 13, ~40,000.

ther trypsin or neuraminidase significantly reduced their mean electrophoretic mobility. Neuraminidase was slightly more effective in the reduction of the mean electrophoretic mobility of amastigotes than trypsin (Table I). DISCUSSION

The amastigote of Trypanosoma cruzi is the only form able to divide within cells of the vertebrate host and is therefore considered responsible for amplification of the acute phase of Chagas disease. However, few data exist on the cell surface properties of this form because it has been difficult to obtain large numbers of amastigotes

without the use of enzymatic treatment during the isolation procedure. Study of the cell surface of amastigotes is of great interest in view of the observation that “a greater number or a higher concentration of antigens recognized by immune sera are expressed on the cell surface of amastigotes than of epimastigotes and trypomastigotes, suggesting that amastigotes may be the stage of T. cruzi better suited to provide antigens for diagnostic, and possibly for immunization, purposes” (Araujo and Remington 1981). Analyzing amastigotes obtained from three different sources and two strains, we concluded that the surface charge, ex-

FIGS. 9- 10. T~panosom~ cruzi amastigotes treated with neuraminidase before incubation in the presence of cationized ferritin particles. In most of the parasites, the binding of the particles to their surface is less intense than that observed in control cells (see Fig. 8). Fig 9, ~32,500; Fig. 10, x 22,500. FIG. 11. T~ypano~oma cruzi amastigote treated with neuraminidase before incubation in the presence of colloidal iron hydroxyde particles. No binding of the particles to the parasite surface is observed. x 22,500.

20

DE CARVALHO,

SOUTO-PADR6N,

AND DE SOUZA

TABLE I Electrophoretic Mobility (EPM) of Amastigotes of T~ypanowma cru~P,~

Source of amastigotes 577408 macrophage Mouse spleen LA9 cells J774G8 macrophage J774G8 macrophage 577468 macrophage

EPM (-urn see-I V-l cm) Strain

Treatment

Mean

SDd

CL Y Y Y Y Y

None None None None TrypsinC NeuraminidaseC

0.86 0.86 0.82 0.85 0.58 0.55

0.10 0.20 0.12 0.02 0.03 0.05

a Data are from 100 cells. b In all instances, I = 0.145 at pH 7.2. ’ Enzymatic treatment as described in Materials and Methods. d Standard deviation.

pressed by the mobility of the cells when submitted to an electric field, is constant and characteristic for this form. Mobility differs from that for Leishmania mexicana amazonensis amastigotes (Pimenta and De Souza 1983). The surface charge of T. cruzi amastigotes is intermediate between that of epimastigotes (-0.65 k 0.11) and that of trypomastigotes (- 1.15 k 0.17 Frn set- ’ V-’ cm). Our previous determination of the mean electrophoretic mobility of the epimastigote and trypomastigote forms (from two different strains and obtained from different sources) indicated that the mean electrophoretic mobility can be used as a criterium to identify the stages of the T. cruzi life cycle (Souto-Padron et al. 1984). Based on determination of adsorptionelution properties on the DEAE-cellulose column, it was previously reported that amastigotes and spheromastigotes of T. cruzi present in old axenic cultures have a surface charge similar to epimastigotes, which have a higher negative surface charge than trypomastigotes (Kreier et al. 1977). Using free-flow electrophoresis, it was recently determined that amastigotes have the lowest charge density, trypomastigotes had an intermediate charge, and epimastigotes the highest charge density (Murray et al. 1982). Our current and former observations (De Souza et al. 1977;

Souto-Padron et al. 1984) indicate that, among parasites suspended in a NaCl solution with an ionic strength of 0.145 mol dmp3, epimastigote and trypomastigote forms have the lowest and the highest negative surface charge, respectively. The charge of amastigotes lies in between. One basic question in the study of the cell surface charge is the determination of the components responsible for the charge. Important information can be obtained by determining the electrophoretic mobility of cells in solutions in which the ionic strength is constant but the pH varies (James 1979). Using this approach, we obtained a graph indicating that the cell surface of T. cruzi amastigotes contains both positively and negatively charged dissociating groups. At low pH, the cell surface is less negative and, after a certain pH (the isoelectric point), the surface becomes positive. At higher pH values, the negative charge increases, probably due to the increase in the dissociation of carboxyl groups, reaching a plateau between pH 7.0 and 10.0 where there is an equilibrium between COO- and NH:. At values above pH 10, the negative charge increases, since in this condition the proton is lost and the charge is due to COO- alone. Studies carried out on various cell types indicate that the negative surface charge is

Trypanosoma

cruzi: AMASTIGOTE SURFACE ANALYSIS

so. 80. 70.

A

60. 50. 40. 30. 20. IO.

80 y

70

= v

60 50 I

B

60. 70.

C

60. 50. 40. 30. LO. IO. 0.4

0.6

0.6

ELECTROPHORETIC

I.0

I.2

14

1.6

I.8

2.0

YOElILITY(-pm.i’.&n)

FIG. 14. Distribution of the electrophoreticmobility

of Trypanosoma cruzi amastigotesisolated from the spleenof infected mice (A), and from the supernatant of LA9 (B) or the macrophage-like(C) cell lines. Fo all measurements,I = 0.145,pH 7.2.

generated by the presence of exposed carboxyl, sulfate, and phosphate groups on the cell surface (Burry and Wood 1979; Eylar et al. 1962; Furghgott and Ponder 1941; Gasic et al. 1963). It has also been shown that sialic acid appears to be a major component contributing to the surface charge (Cook et al. 1961; Eylar et al. 1962; Seaman and Uhlenbruck 1963; Seaman and Heard 1960). Treatment with trypsin or neuraminidase reduces the electrophoretic mobility of T. cruzi amastigotes. Although we do not know which components of the cell surface are released by trypsin treatment, the results obtained with neuraminidase indicate that sialic acid, sensitive to the enzyme iso-

21

lated from Clostridium perfringens, is exposed on the surface of amastigotes of T. cruzi and accounts for about 37% of the negative surface charge. Previous studies have shown that lectins which interact with sialic acid, such as wheat germ agglutinin (WGA) and the lectin from Limulus polyphemus, induced agglutination only when used at very high concentrations (Pereira et al. 1980). These observations led the authors to suggest that T. cruzi amastigotes are the least sialized forms in the T. cruzi life cycle. Our observations, however, indicate that amastigotes have a more negative surface charge than epimastigotes and that neuraminidase treatment reduces the surface charge of amastigotes by 37%. Our previous observations showed that neuraminidase treatment reduced the electrophoretic mobility of epimastigote and trypomastigote forms by 18 and 50%, respectively (Souto-Padron et al. 1984). Also, our cytochemical observations show the presence of sialic acid on the surface of T. cruzi amastigotes. It has been indicated previously that, at low pH values such as pH 1.8 (usually employed with colloidal iron hydroxyde), only certain groups (mainly sialic acid) are exposed (Gasic et al. 1963). We observed that treatment of amastigotes with neuraminidase abolished or significantly reduced the binding of colloidal iron and cationized ferritin particles to the parasite’s surface. Our observations on freeze-fracture replicas of the isolated amastigotes show that both faces of their plasma membrane have a density of intramembranous particles which is slightly higher for trypomastigotes (protoplasmic face, 122 IMP/pm*) and much lower for epimastigotes (protoplasmic face, 1830 IMP/pm2) (De Souza et al. 1978). The particles were homogenously distributed, with the exception of the region of the cytostome where there is an area delimited by a pallisade of adjacent particles. This structure is basically similar to that seen in epimastigotes (Martinez-Palomo et

22

DE CARVALHO,

SOUTO-PADRGN,

AND DE SOUZA

FIG. 15. Influence of the pH of the solution in which the Trypalzd~oma cruzi amastigotes are suspended on their electrophoretic mobility.

al. 1976; De Souza et al. 1978). However, while the membrane which lines the cytostome of epimastigotes has few particles, the one lining the cytostome of amastigotes has many particles. All studies on the biology of the rounded forms of Trypanosoma cruzi, which have received various designations such as leishmanoids, amastigote-like forms, spheromastigote, micromastigote, and staphylomastigote, have been with parasites outside the vertebrate host cell (Brener 1973; De Souza 1984). However, up to now there were no markers with which to test the possibility that the rounded forms are equivalent to those which multiply within the vertebrate cells. It is possible that the mean electrophoretic mobility can be a useful marker for identifying amastigotes of Trypanosoma

cvuzi. ACKNOWLEDGMENTS

We thank Dr. H. Meyer for advice and suggestions in the preparation of this manuscript. The work has been supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq), Conselho de Ensino para Graduados da UFRJ (CEPG), and Financiadora de Estudos e Projectos (FINEP). REFERENCES I. A., KATZIN, A. M., AND MILDER, R. V 1983. A method for isolation Trypanosoma

ABRAHAMSON,

cruzi amastigotes from spleen and liver using twostep discontinuous gradient centrifugation. Journal of Parasitology 68, 437-439. ARA~JO, F. G., AND REMINGTON, J. S. 1981. Characterization of stages and strains of Trypunosoma cruzi by analysis of cell membrane components. Journal of Immunology 127, 855-859. BRENER, Z. 1973. Biology of Trypanosomn cruzi. Annual Review of Microbiology 27, 347-382. BURRY, R. W., AND WOOD, J. G. 1979. Contributions of lipids and proteins to the surface charge of membranes. Journal of Cell Biology 82, 726-741. CARVALHO, R. M. G., MEIRELLES, M. N. L., DE SOUZA, W., AND LEON, W. 1981. Isolation of the intracellular stage of Trypanosoma cruzi and its interaction with mouse macrophages in vifro. Infection and Immunity 33, 546-554. COLLI,

W., ANDREWS,

N. W., AND ZINGALES,

B. 1981.

Surface determinants in American trypanosomes. In “International Cell Biology” (H. C. Schweiger, ed.), pp. 401-410. Springer-Verlag, BerliniHeidelberg/New York. COOK, G. M. W., HEAD, D. H., AND SEAMAN, G. V. E 1961. Sialic acid and the electrokinetic charge of the human erythrocytes. Nature (London) 191, 44-47. DANON, D., GOLDSTEIN, L., MARIKOVSKY, SKUTELSKY, E. 1972. Use of cationized

Y., AND

ferritin as a label of negative charges on cell surface. Journal of Ultrastructure Resenrch 38, 500-510. DE CARVALHO, T. U., AND DE SOUZA, W. 1983. Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells. Zeitschrift fur Parasitenkunde 69, 571-575, DE SOUZA, W. 1984. Cell Biology of Trypanosoma cruzi. International Review of Cytology 86, 197283. DE SOUZA: W., ARGUELLO, C., MARTINEZ-PALOMO, A., TRISSL, D., GONZALES-ROBLES,

A., AND CHIARI,

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E. 1977. Surface charge of Trypunosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility. Journal of Protozoology 24, 41l-415. DE SOUZA, W., MARTINEZ-PALOMO, A., AND GONZALES-ROBLES,A., 1978. The cell surface of Ttypanosoma cruzi: Cytochemistry and freeze-fracture. Journal of Cell Science 33, 285-299. EYLAR, E. H., MADOFF, M. A., BRODY, 0. V., AND ONCLEY, S. L. 1962. The contribution of sialic acid to the surface charge of the erythrocyte. Journal of Biological Chemistry 237, 1192-2006. FURGHGOTT, R. F., AND PONDER,E. 1941. Electrophoretie studies on human red blood cells. Journal of General Physiology 24, 447-457. GASIC,G. J., BERWICK,L., AND SORRENTINO, M. 1968. Positive and negative colloidal iron as cell surface electron stains. Laboratory Investigation 18, 63-71. GUTTERIDGE,W. E., COVER, B., AND GABORAK, M. 1978. Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism. Parasitology 76, 159- 176. JAMES, A. M. 1979. Molecular aspects of biological surfaces. Chemical Society Review 8, 389-418. KREIER, J. P., AL-ABBASSY, S. N., AND SEED, T. M. 1977. Trypanosoma cruzi. Surface charge characteristics of cultured epimastigotes, trypomastigotes and amastigotes. Revista do Institute de Medicina Tropical de S&o Paula 19, 10-20. MARTINEZ-PALOMO, A., DE SOUZA, W., AND GONZALES-ROBLES,A. 1976. Topographical differences in the distribution of surface coat components and intramembranous particles. A cytochemical and freeze-fracture study in culture forms of Trypanosoma cruzi. Journal of Cell Biology 69, 507-513.

SURFACE ANALYSIS

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