Interaction With the Extracellular Matrix Triggers Calcium Signaling in Trypanosoma cruzi Prior to Cell Invasion.

Trypanosoma cruzi, the etiological agent of Chagas disease in humans, infects a wide variety of vertebrates. Trypomastigotes, the parasite infective forms, invade mammalian cells by a still poorly understood mechanism. Adhesion of tissue culture- derived trypomastigotes to the extracellular matrix (ECM) prior to cell invasion has been shown to be a relevant part of the process. Changes in phosphorylation, S-nitrosylation, and nitration levels of proteins, in the late phase of the interaction (2 h), leading to the reprogramming of both trypomastigotes metabolism and the DNA binding profile of modified histones, were described by our group. Here, the involvement of calcium signaling at a very early phase of parasite interaction with ECM is described. Increments in the intracellular calcium concentrations during trypomastigotes-ECM interaction depends on the Ca2+ uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca2+ channels and sphingosine-dependent plasma membrane Ca2+ channel, but not by Vanadate, an inhibitor of the plasma membrane Ca2+-ATPase. Furthermore, Nifedipine inhibits the invasion of host cells by tissue culture- derived trypomastigotes in a dose-dependent manner, reaching 95% inhibition at 100 µM Nifedipine. These data indicate the importance of both Ca2+ uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca2+ uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca2+ homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca2+ concentration in the presence of Antimycin A, in contrast to other calcium homeostasis disruptors, such as Cyclopiazonic acid for endoplasmic reticulum and Bafilomycin A for acidocalcisome. Total phosphatase activity in the parasite decreases in the presence of Nifedipine, EGTA, and Okadaic acid, implying a role of calcium in the phosphorylation level of proteins that are interacting with the ECM in tissue culture- derived trypomastigotes. In summary, we describe here the increment of Ca2+ at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca2+ channels in the influx of Ca2+ and the mitochondrion as the relevant organelle in Ca2+ homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.

Reprogramming of Trypanosoma cruzi metabolism triggered by parasite interaction with the host cell extracellular matrix.

Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis.

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, ß-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.

Down regulation of NO signaling in Trypanosoma cruzi upon parasite-extracellular matrix interaction: changes in protein modification by nitrosylation and nitration.

BACKGROUND: Adhesion of the Trypanosoma cruzi trypomastigotes, the causative agent of Chagas' disease in humans, to components of the extracellular matrix (ECM) is an important step in host cell invasion. The signaling events triggered in the parasite upon binding to ECM are less explored and, to our knowledge, there is no data available regarding •NO signaling. METHODOLOGY/PRINCIPAL FINDINGS: Trypomastigotes were incubated with ECM for different periods of time. Nitrated and S-nitrosylated proteins were analyzed by Western blotting using anti-nitrotyrosine and S-nitrosyl cysteine antibodies. At 2 h incubation time, a decrease in NO synthase activity, •NO, citrulline, arginine and cGMP concentrations, as well as the protein modifications levels have been observed in the parasite. The modified proteins were enriched by immunoprecipitation with anti-nitrotyrosine antibodies (nitrated proteins) or by the biotin switch method (S-nitrosylated proteins) and identified by MS/MS. The presence of both modifications was confirmed in proteins of interest by immunoblotting or immunoprecipitation. CONCLUSIONS/SIGNIFICANCE: For the first time it was shown that T. cruzi proteins are amenable to modifications by S-nitrosylation and nitration. When T. cruzi trypomastigotes are incubated with the extracellular matrix there is a general down regulation of these reactions, including a decrease in both NOS activity and cGMP concentration. Notwithstanding, some specific proteins, such as enolase or histones had, at least, their nitration levels increased. This suggests that post-translational modifications of T. cruzi proteins are not only a reflex of NOS activity, implying other mechanisms that circumvent a relatively low synthesis of •NO. In conclusion, the extracellular matrix, a cell surrounding layer of macromolecules that have to be trespassed by the parasite in order to be internalized into host cells, contributes to the modification of •NO signaling in the parasite, probably an essential move for the ensuing invasion step.

The Gp85 surface glycoproteins from Trypanosoma cruzi.

Trypanosoma cruzi strains show distinctive characteristics as genetic polymorphism and infectivity. Large repertoires of molecules, such as the Gp85 glycoproteins, members of the Gp85/Trans-sialidase superfamily, as well as multiple signaling pathways, are associated with invasion of mammalian cells by the parasite. Due to the large number of expressed members, encoded by more than 700 genes, the research focused on this superfamily conserved sequences is discussed. Binding sites to laminin have been identified at the N-terminus of the Gp85 molecules. Interestingly, the T. cruzi protein phosphorylation profile is changed upon parasite binding to laminin (or fibronectin), particularly the cytoskeletal proteins such as those from the paraflagellar rod and the tubulins, which are both markedly dephosphorylated. Detailed analysis of the signaling cascades triggered upon T. cruzi binding to extracellular matrix (ECM) proteins revealed the involvement of the MAPK/ERK pathway in this event. At the C-terminus, the conserved FLY sequence is a cytokeratin-binding domain and is involved in augmented host cell invasion in vitro and high levels of parasitemia in vivo. FLY, which is associated to tissue tropism and preferentially binds to the heart vasculature may somehow be correlated with the severe cardiac form, an important clinical manifestation of chronic Chagas' disease.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells.

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other α-aminocarbonyl metabolites, DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H(2)O(2), NH(4)(+) ion, and a highly toxic α-oxoaldehyde. In vitro, DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC(50)c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 µM) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 µM buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells.

Adhesion of Trypanosoma cruzi trypomastigotes to fibronectin or laminin modifies tubulin and paraflagellar rod protein phosphorylation.

BACKGROUND: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. CONCLUSIONS/SIGNIFICANCE: Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Vesicles as carriers of virulence factors in parasitic protozoan diseases.

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell-cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation.

The α-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37°C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other α-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml-1) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(•), and those with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(•) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 µM) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity.

Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo.

BACKGROUND: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature. METHODS: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied. FINDINGS AND CONCLUSIONS: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

Glucose uptake in the mammalian stages of Trypanosoma cruzi.

Trypanosoma cruzi, the agent of Chagas' disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V(max)=5.34+/-0.54 nmol/min per mg of protein; K(m)=0.38+/-0.01 mM) when compared to intracellular epimastigotes (V(max)=2.18+/-0.20 nmol/min per mg of protein; K(m)=0.39+/-0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.

Role of the gp85/trans-sialidase superfamily of glycoproteins in the interaction of Trypanosoma cruzi with host structures.

Invasion of mammalian cells by T. cruzi trypomastigotes is a multi-step and complex process involving several adhesion molecules, signaling events and proteolytic activities. From the blood to the cell target in different tissues the parasite has to interact with different cells and the extracellular matrix (ECM). The review focus on the role of the gp85/ trans-sialidase superfamily members in the interaction of the parasite with the host cell, particularly with ECM components, with emphasis on the significant variability among the ligands and receptors involved. Use of the SELEX technique to evolve nuclease-resistant RNA aptamers for receptor identification is briefly discussed.

Trypanosoma cruzi: adhesion to the host cell and intracellular survival.

Both invasion of the host cell by T. cruzi and its establishment into the mammalian host are critical steps. In this review, the adhesion step and the intracellular survival in non-professional phagocytes are particularly focused on, with special emphasis on the role of Gp85/trans-sialidase (Gp85/TS) superfamily. Excellent reviews have been published lately, some covering other aspects of T. cruzi-host interaction and will be cited instead of the original articles due to limited number of listed references.

Enucleated L929 cells support invasion, differentiation, and multiplication of Trypanosoma cruzi parasites.

Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection.

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi.

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.

Aspartate transport and metabolism in the protozoan parasite Trypanosoma cruzi.

Aspartate is one of the compounds that induce the differentiation process of the non-infective epimastigote stage to the infective trypomastigote stage of the protozoan parasite Trypanosoma cruzi. l-aspartate is transported by both epimastigote and trypomastigote cells at the same rate, about 3.4 pmolmin(-1) per 10(7) cells. Aspartate transport is only competed by glutamate suggesting that this transport system is specific for anionic amino acids. Aspartate uptake rates increase along the parasite growth curve, by amino acids starvation or pH decrease. The metabolic fate of the transported aspartate was predicted in silico by identification of seven putative genes coding for enzymes involved in aspartate metabolism that could be related to the differentiation process.

Tempol diverts peroxynitrite/carbon dioxide reactivity toward albumin and cells from protein-tyrosine nitration to protein-cysteine nitrosation.

Tempol has been shown to protect experimental animals from injuries associated with excessive nitric oxide production. In parallel, tempol decreased the levels of protein-3-nitrotyrosine in the injured tissues, suggesting that it interacted with nitric oxide-derived oxidants such as nitrogen dioxide and peroxynitrite. Relevantly, a few recent studies have shown that tempol catalytically diverts peroxynitrite/carbon dioxide reactivity toward phenol from nitration to nitrosation. To examine whether this shift occurs in biological environments, we studied the effects of tempol (10-100 microM) on peroxynitrite/carbon dioxide (1 mM/2 mM) reactivity toward proteins, native bovine serum albumin (BSA) (0.5-0.7 cys/mol) and reductively denatured BSA (7-19 cys/mol), and cells (J774 macrophages). Although not a true catalyst, tempol strongly inhibited protein-tyrosine nitration (70-90%) and protein-cysteine oxidation (20-50%) caused by peroxynitrite/carbon dioxide in BSA, denatured BSA, and cells while increasing protein-cysteine nitrosation (200-400%). Tempol consumption was attributed mainly to its reaction with protein-cysteinyl radicals. Most of the tempol, however, reacted with the radicals produced from peroxynitrite/carbon dioxide, that is, nitrogen dioxide and carbonate radical anion. Accordingly, tempol decreased the yields of BSA-cysteinyl and BSA-tyrosyl/tryptophanyl radicals, as well their decay products such as protein-3-nitrotyrosine. The parallel increase in protein-nitrosocysteine yields demonstrated that part of the peroxynitrite is oxidized to nitric oxide by the oxammonium cation produced from tempol oxidation by peroxynitrite/carbon dioxide-derived radicals. Protein-nitrosocysteine formation was shown to occur by radical and nonradical mechanisms in studies with a protein-cysteinyl radical trapper. These studies may contribute to the understanding of the protective effects of tempol in animal models of inflammation.

Modeling the Trypanosoma cruzi Tc85-11 protein and mapping the laminin-binding site.

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.

Post genomic analysis of permeases from the amino acid/auxin family in protozoan parasites.

The "amino acid/auxin permeases" is probably the most represented family of transporters in the Trypanosoma cruzi genome. Using a high-throughput searching routine and preliminary data from the T. cruzi genome project, more than 15,000 sequences were iteratively assembled into contigs, and 60 open reading frames corresponding to different putative amino acid transporters, clustered in 12 groups, were detected and characterized in silico. T. cruzi genomic organization of such sequences showed that these putative amino acid transporter genes are in an unusually large number and arranged in repeat clusters comprising about 0.2% of the genome. These data suggest that the family has evolved following tandem duplication events and constitutes a novel family of variable proteins in protozoan organisms. The mRNA expression of the predicted genes was demonstrated in infective and non-infective parasite forms. Orthologous sequences were also identified in other unicellular parasites such as Leishmania spp., Plasmodium spp., and Trypanosoma brucei.