Microbicidal property of B1 cell derived mononuclear phagocyte.

A mononuclear phagocyte derived from B1b cells (B1CDP) has been described. As these cells migrate from the peritoneal cavity to non-specific inflammatory lesion sites and are highly phagocytic via Fc and mannose receptors, their microbicidal ability of these cells was investigated using the Coxiella burnetii cell infection model in vitro. In this report, the pattern of infection and C. burnetii phase II survival in B1CDP phagosomes was compared with the pattern of infection of peritoneal macrophages from Xid mice (PMphi) and bone marrow derived macrophages (BMMphi). Infection was assessed by determining the large parasitophorous vacuole formation, the relative focus forming units and the quantification of DAPI (4',6-diamino-2-phenylindole) fluorescence images acquired by confocal microscopy. When compared to macrophages, B1CDP are more permissive to the bacterial infection and less effective to kill them. Further, results suggest that IL-10 secreted by B1 cells are involved in their susceptibility to infection by C. burnetti, since B1CDP from IL-10 KO mice are more competent to control C. burnetii infection than cells from wild type mice. These data contribute further to characterize B1CDP as a novel mononuclear phagocyte.

The distribution of motor proteins in the muscles and flame cells of the Schistosoma mansoni miracidium and primary sporocyst.

Schistosoma mansoni eggs, miracidia and primary sporocysts were labelled with phalloidin-rhodamine to visualize filamentous actin structures. Analysis of these forms by confocal fluorescence microscopy revealed the presence of previously well-defined circular and longitudinal muscle layers. Besides these muscular layers that sustain and provide motility to these parasite forms, we found in these 3 consecutive developmental stages of the parasite previously unidentified actin-rich tubular structures. In the 3 forms, 4 actin-rich tubules could be observed by optical sectioning underneath the well-developed muscle layers. The tubules appear in pairs, transversal to the length of the parasite, and located towards the extremities. By using an anti-flame cell specific antibody we confirmed that the tubules co-localize with flame cells and also determined that the tubule core is filled with microtubules. The additional presence of myosin in these tubules strongly suggests that they are contractile structures.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection.

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.

Comparative histopathology of endomyocardial biopsies in chagasic and non-chagasic heart transplant recipients.

BACKGROUND: Heart transplantation has been an option for the treatment of chagasic (C) cardiomyopathy despite difficulties concerning the control of rejection and reactivation. The parasite-host interaction under the influence of immunosuppressive therapy may affect the immunological response to the graft in a pattern different from that in non-chagasic (NC) patients. The aim of this study was to compare the major histopathological features in heart transplantation in C and NC patients. METHODS: We studied 293 endomyocardial biopsies from two groups of heart transplanted patients, including 18 C and 15 NC. Both groups had identical surgical and clinical procedure except immunosuppressive therapy was lower in C patients. The histopathological parameters evaluated were the Quilty effect, rejection, C myocarditis reactivation, fibrosis, hypertrophy, and ischemia. In addition, lymphocytic cellular infiltration of myocarditis due to rejection or reactivation was immunophenotyped in the biopsies of both groups with rejection grades 3 to 4, in biopsies with signs of reactivation, and in fragments of the receptor heart with chronic C myocarditis. A search for Trypanosoma cruzi was performed in all biopsies in the C group in which lymphocyte immunophenotyping was done. We used immunofluorescence and confocal microscopy. RESULTS: The Quilty effect was present in 23% of the biopsies, involving 69.7% of the patients without a significant difference between groups (p = 0.509). Rejection was frequently observed in biopsies with the Quilty effect and the effect often recurred in the same patient. Rejection grades 3 to 4 was more frequent in the C group (p = 0.023). There were 5 episodes of Chagas' disease reactivation with myocarditis in 2 cases. The mean numbers of CD8+ and CD4+ T cells, and the CD4+-to-CD8+ ratio were similar for rejection in both groups (p > 0.05), while the CD4+-to-CD8+ ratio was significantly lower in chronic C myocarditis compared to rejection in the C group (p = 0.043). There was no significant difference in ischemic damage or interstitial fibrosis in the groups but there was a higher frequency of hypertrophy in the NC group (p = 0.007). CONCLUSIONS: The histopathological features of heart transplantation in C patients did not differ from that in NC patients in regard to the Quilty effect, development of myocardial fibrosis and ischemia. However, the higher involvement of the C group for rejection grades 3 to 4 suggested higher susceptibility to this event. The similarity of the lymphocytic cellular composition for rejection in both groups indicates that C patients respond to immunological stimulus in a similar pattern as NC patients.

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation.

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.

Phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes.

Trypanosoma cruzi trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.

In vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins.

Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization.

Study of acute chagasic mice under immunosuppressive therapy by cyclosporin A : modulation and confocal analysis of inflammatory reaction.

The in vivo effects of cyclosporin A (CsA) on Trypanosoma cruzi infection were examined using different schedules of the drug in mice infected with the Y strain. Parasitaemia at day 8 after infection among CsA-treated animals was usually higher than control infected non-treated mice. On the other hand, mortality analysis showed that animals CsA-treated either with 200 mg/kg 2 days before infection or with therapeutic doses (10 mg/kg every other day) showed almost the same mean time of death (35.8 and 38.2 days, respectively). In these groups mice died 50% less than control infected non-treated ones. The mean time of death in the animals treated with 200 mg/kg 5 days after infection and in infected non-treated control mice were respectively 29.0 and 22.6 days. The kinetics analysis of the leukocyte population of animals treated with a single dose of 200 mg/kg of CsA before or after infection did not show the alternate pattern of leukopenia/leukocytosis observed in control groups of infected mice but differential cell counts indicated a modulatory action upon circulating leukocytes of therapeutic doses of CsA. The animals treated with any of the CsA schedules showed a moderate to intense diffuse inflammatory reaction exhibiting mainly mononuclear cells in the heart. Immunofluorescence analysis by confocal microscopy revealed that macrophages are a major component of the inflammatory infiltrate in all groups of CsA-treated mice and also in the control group.

Trypanosoma cruzi: amastigote polymorphism defined by monoclonal antibodies

We have raised monoclonal antibodies (mAbs) directed towards amastigote forms of Trypanosoma cruzi, and shown that mAbs 1D9 and 4B9 are carbohydrate while mAb 4B5 activity is resistant to periodate oxidation of the antigen. Here we used an ELISA to quantitate and compare the expression of surface epitopes on fixed parasites among different parasite isolates. The expression of markers varied among T. cruzi amastigotes isolated from infected cells or after extracellular differentiation of trypomastigotes. Moreover, we also observed an extensive polymorphic expression of these epitopes among amastigotes derived from different strains and clones. For instance, mAb 2C2 strongly and evenly reacted with 9 strains and clones (G, Y, CL, Tulahuen, MD, and F, and clones Sylvio X-10/4, D11, and CL.B), with absorbance at 492 nm (A492 nm) from 0.6 to 0.8. By contrast, mAb 4B5 had a higher expression in Tulahuen amastigotes (around 0.9 at 492 nm) whereas its reactivity with amastigotes from clones CL.B, Sylvio X-10/4 and D11 was much lower (around 0.4). mAb 1D9 displayed an interesting pattern of reactivity with amastigotes of the different strains and clones (A492 nm of G>D11ÝSylvio X-10/4 = MD>Tulahuen = F = Y>CL>CL.B). Finally, we observed that mAb 4B9 had the lowest reaction with the parasites studied, with higher values of A492 nm with Y strain (around 0.6) and lower values with Tulahuen, F and CL.B strains (around 0.2). Immunoblotting analysis also showed extensive variations among amastigotes of the various parasite isolates and mAbs 4B9, 1D9 and 4B5 revealed significant differences in expression between clones and parental strains. These data describe a previously uncharacterized polymorphism of T. cruzi amastigote surface components

Trypanosoma cruzi: effect of protein kinase inhibitors and cytoskeletal protein organization and expression on host cell invasion by amastigotes and metacyclic trypomastigotes.

Although trypomastigotes are regarded as the classic infective forms of T. cruzi, amastigotes generated extracellularly or released from infected cells during lysis may circulate and infect other cells. We have compared the infectivity of metacyclic trypomastigotes and extracellular amastigotes toward HeLa and Vero cells and observed that amastigotes were capable of invading both HeLa and Vero cells to a much higher degree than the corresponding metacyclic forms. Second, cell microfilament or microtubule disruption inhibited amastigote but not trypomastigote entry. Third, cells with altered expression in cytoskeletal components (ABP or gelsolin) internalize amastigotes and trypomastigotes with highly contrasting fashion. Fourth, protein kinase inhibitors such as genistein and staurosporine affect the internalization of amastigotes and trypomastigotes in a host-cell-dependent manner. Our results suggest that extracellular amastigotes and metacyclic trypomastigotes utilize mechanisms to invade host cells with particular features for each T. cruzi form and for each host cell. When internalized, both forms associate to lysosomes of HeLa cells.

Axenic cultivation and partial characterization of Leishmania braziliensis amastigote-like stages.

Leishmania braziliensis strain M2903 was adapted for growth and serially maintained as amastigotes at 34 degrees C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, amastigote growth took place in the absence of supplementary haemin and was unaffected when the initial medium pH was adjusted between 5.4 and 6.3. In contrast to promastigotes, which were elongated and exhibited very long free flagella endowed with the paraflagellar rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in axenic amastigotes was confirmed in Western blots and confocal immunofluorescence microscopy, by lack of reactivity with mAb 1B10. The antibody, which specifically labelled the paraflagellar structure, recognized a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes and two 70/74 kDa related proteins in L. braziliensis promastigotes. Surface 125I-labelling experiments identified promastigote-specific components (> 100, 74, 45/47 and 28 kDa) and at least 1, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respectively at 50 and 12.5 micrograms/ml, promastigotes were not agglutinated by PNA and agglutinated in the presence of LCA at concentrations of 100 micrograms/ml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerous organisms, able to proliferate at 34 degrees C in UM-54 medium, could be recovered 48 h later.

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.

Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera.

Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.

An association between actin and nucleocapsid polypeptides in isolated murine retroviral particles.

Mammalian cells infected with retroviruses frequently display virus particles budding at the tip of cellular projections resembling microvilli and filopodia. In normal and infected cells these cellular projections contain actin microfilaments and in specialized retrovirus-tipped projections from the P815 cell, a direct association between actin filaments and the apical virus particle could be demonstrated (Mortara and Koch, 1986). Here we confirm and extend these observations using a murine macrophage cell line chronically infected with a C-type retrovirus. Immunochemical and biochemical methods were used to identify actin-associated and actin-binding components among the retroviral polypeptides. The results show that Pr65gag and its p15 N-terminal domain can bind to actin in vitro and may be major binding sites for actin filaments on the retroviral nucleocapsid.

Studies on trypanosomatid actin. I. Immunochemical and biochemical identification.

In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.

Endooligopeptidase A activity in rabbit heart: generation of enkephalin from enkephalin containing peptides.

Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met5] enkephalin and [Leu5]enkephalin from small enkephalin containing peptides. They also hydrolyze the Phe5-Ser5 bond of bradykinin and the Arg8-Arg9 bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr = 73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr = 72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulin reacts with a polypeptide corresponding to Mr = 50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.

Reactivity of stage-specific monoclonal antibody 1G7 with metacyclic trypomastigotes of Trypanosoma cruzi strains: lytic property and 90,000 mol. wt surface antigen polymorphism.

Eleven strains of Trypanosoma cruzi, originating from a variety of vertebrate and invertebrate hosts in distinct geographical regions, were examined for the reactivity of metacyclic stages with the monoclonal antibody 1G7. Trypomastigotes of five strains were susceptible to complement-dependent 1G7-mediated lysis. Higher levels of 1G7 bound to metacyclics of lysis-susceptible strains as compared to lysis-resistant isolates. Excluding Y and CL strains, 1G7 reacted with metacyclics of all T. cruzi isolates by binding to a 90,000 mol. wt surface polypeptide. A 90,000 mol. wt protein lacking the 1G7-specific epitope but immunologically related to the 90,000 mol. wt antigen of other T. cruzi isolates is present in Y and CL strains. The intensity of the 90,000 mol. wt band, detected by surface iodination of metacyclics or in immunoblots using the monoclonal antibody 1G7 or the monospecific antiserum to 90,000 mol. wt protein, varied among different strains and also a discrete variation was observed in its molecular weight. The overall analysis reveals a polymorphism of the 90,000 mol. wt protein, which is ubiquitous among the different T. cruzi isolates.

Analysis of pseudopodial structure and assembly with viral projections.

The mechanisms by which cells extend motile pseudopodial projections are still poorly understood. Several fundamental mechanisms have been proposed on the basis of hydrostatic pressure, membrane addition and microfilament reorganization. A common focus of all such mechanisms is the growing tip of a pseudopodium. Yet some basic questions about the nature of the tip in natural pseudopodia remain obscure. However, one class of structure, the virus-tipped projections, often contains a well-defined particle, both morphologically and biochemically, and therefore provides a useful model system for the examination of the tips of cellular projections. In P815 cells the virus-tipped projections are long, thin structures closely resembling filopodia in other cells. The apical virus particle is a retrovirus particle produced by the chronic infection existing in this cell line. In demembranated filopodia, the virus particle retains a tight association with a single actin microfilament. Biochemical analyses indicate that the major retroviral structural polypeptide Pr65 is an actin-binding protein that could provide the anchorage site for the actin filament. The existence of a solid virus particle tethered by an actin filament to the cytoskeleton makes it very unlikely that these projections grow by membrane addition at the tip. The major positive implication is that the apex of a projection does not relinquish its interaction with the submembranous cytoskeleton during growth. Such an arrangement would be compatible with either a hydrostatic-pressure-driven or a cytoskeleton-driven mechanism of filopodial growth.