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W de Souza

Publications and source records attributed to W de Souza.

At least 37 records · Page 2Linked to original sources
PubMed2001

Cloning and characterization of the metacyclogenin gene, which is specifically expressed during Trypanosoma cruzi metacyclogenesis.

We isolated a gene that is differentially expressed during Trypanosoma cruzi metacyclogenesis by the representation of differential expression (RDE) method, using differentiating epimastigotes cultured in chemically defined medium. This gene, the metacyclogenin gene, encodes a 630-nucleotide mRNA that is specifically associated with the polysomes of epimastigotes allowed to differentiate for 24 h. We sequenced and characterized the metacyclogenin gene and found that there were at least three copies of the gene organized into tandem 2.8 kb repeats in the genome of T. cruzi Dm28c. We analyzed the repeats and found that they contained two other genes, one encoding tryparedoxin peroxidase and the other encoding a 0.6 kb mRNA (named associated gene or AG) with sequences showing no significant similarity to those in the GenBank database. Northern blot analysis of polysomal RNA extracted from replicating and differentiating epimastigotes showed that metacyclogenin and AG genes displayed similar patterns of expression. Their products were detected only in differentiating epimastigotes, whereas tryparedoxin peroxidase was detected only in the polysomal RNA fraction of replicating and differentiating epimastigotes. In Northern blots of total RNA from differentiating and replicating epimastigotes, the genes studied were detected in both cell populations. The differential expression of the metacyclogenin gene was confirmed by immunocytochemistry studies showing that the protein is detected only in differentiating (adhered) epimastigote. The results suggest that mRNA mobilization to polysomes is an important mechanism in the regulation of gene expression in T. cruzi.

Amino Acid Sequence↗
PubMed2001

Evidence that increased tyrosine phosphorylation causes disassembly of adherens junctions but does not perturb paracellular permeability in Caco-2 cells.

In this study, we report on the apparent effect of increased tyrosine phosphorylation events on the assembly and integrity of adherens junctions (AJs) and on paracellular permeability in Caco-2 cells. Cell monolayers were incubated with the phosphotyrosine phosphatase inhibitor vanadate/H2O2. Addition of this compound to monolayer resulted in disruption of the AJs, as revealed by electron microscopy and by a loss of membrane association of the AJ-associated protein uvomorulin/E-cadherin (U/E-c). However, tight junctions (TJs) were unaltered, as determined by measuring the transepithelial resistance (Rt), by ruthenium red labeling, as seen by transmission electron microscopy, and the distribution of TJ strands as seen in freeze-fracture replicas and by hyperphosphorylation of triton-insoluble occludin. Also examination of vanadate/H2O2 treated cells indicated a specific increase in AJ-associated phosphotyrosine residues as evaluated by immunofluorescence microscopy, but no modification of F-actin distribution, as revealed by confocal laser scanning microscopy analysis. To verify that modulation of AJs was indeed related to tyrosine phosphorylation, we tested a range of distinct protein kinase inhibitors. Of the three inhibitors tested (tyrphostin 25, genistein and staurosporine), tyrphostin 25 completely blocked the effects of vanadate/ H2O2 on assembly and integrity of AJs, redistribution of U/E-c and phosphotyrosine labeling. Our results indicate that, after addition of vanadate/H2O2 to Caco-2 monolayers, specific tyrosine phosphorylation of proteins cause disruption of AJs, but no modifications of the TJs' structure and functionality. These observations suggest that, in contrast to what happens with epithelial cells, TJs and AJs of Caco-2 cells are regulated by independent mechanisms.

Actins↗
PubMed2000

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus.

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.

Animals↗
PubMed2000

Phagocytosis of yeast: a method for concurrent quantification of binding and internalization using differential interference contrast microscopy.

In studies of phagocytosis there is a need to distinguish targets that are internalized by the cell from those that are bound to the cell surface. The present work describes a simple method by which internalized and surface-bound yeast particles can be identified by differential interference contrast microscopy, using trypan blue to stain surface-bound yeast particles. The method has the advantage that both internalized and surface-bound particles can be visualized without the need to switch the illumination source and/or filter sets, thus facilitating concurrent quantitation of binding and internalization. The method was evaluated with the phagocytosis-modulating agents horseradish peroxidase (HRP) and cytochalasin D, using adherent resident macrophages as phagocytic cells. When macrophages are challenged with a particular type of target, they usually bind many more targets than they ingest. It was shown that yeast particles were arrested in the initial binding phase of phagocytosis depending on the region of macrophage plasma membrane where binding sites were formed. Failure of surface-bound yeast particles to trigger internalization was not due to modifications of the yeast particle surface. Nor was it due to binding to non-phagocytic receptors, or low-affinity receptor-ligand interactions. The glycoprotein HRP inhibited only the binding stage of phagocytosis, whereas cytochalasin D, a drug that affects actin polymerization, inhibited both binding and internalization. However, when the yeast particles were pre-incubated in fresh mouse serum, cytochalasin D inhibited only the internalization step. The assay described here may be useful in studies concerned with the function and expression of phagocytosis-mediating surface lectins.

Animals↗
PubMed2000

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication.

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.

Animals↗
PubMed2000

Evidence for a trypanothione-dependent peroxidase system in Trypanosoma cruzi.

Hydroperoxide metabolism in Crithidia fasciculata has recently been shown to be catalyzed by a cascade of three oxidoreductases comprising trypanothione reductase (TR), tryparedoxin (TXN1), and tryparedoxin peroxidase (TXNPx) (Nogoceke et al., Biol. Chem. 378, 827-836, 1997). The existence of this metabolic system in the human pathogen Trypanosoma cruzi is supported here by immunohistochemistry. Epimastigotes of T. cruzi display strong immunoreactivity with antibodies raised against TXN1 and TXNPx of C. fasciculata. In addition, a full-length open reading frame presumed to encode a peroxiredoxin-type protein in T. cruzi (Acc. Nr. AJ 012101) was heterologously expressed in Escherichia coli and shown to exhibit tryparedoxin peroxidase activity. With TXN, TXNPx, trypanothione and TR, T. cruzi possesses all components constituting the crithidial peroxidase system. It is concluded that the antioxidant defense of T. cruzi also depends on the NADPH-fuelled, trypanothione-mediated enzymatic hydroperoxide metabolism.

Amino Acid Sequence↗
PubMed2000

Characterization of a Rab11 homologue in Trypanosoma cruzi.

Vesicle trafficking between organelles occurs through fusion of donor and specific acceptor membranes. This process is highly regulated and ensures proper direction in sorting and packaging of a number of molecules in eukaryotic cells. Monomeric GTPases of the Rab family play a pivotal role in the control of membrane fusion and vesicle traffic. In this paper, we characterize a Trypanosoma cruzi Rab 11 homologue (TcRab11) that shares at, the amino acid level, 40% similarity with human rab11, Arabdopsis thaliana rab11 and yeast rab11 homologue genes. Western blot analysis, using a polyclonal rabbit antiserum raised against a synthetic peptide derived from the COOH-terminus of predicted the TcRab11 protein, reacted to a 26kDa protein. In immunofluorescence assays, TcRab 11, was shown to be expressed in epimastigote and amastigote forms, but it was absent in trypomastigotes. Interestingly, the TcRab11 product seems to be located at the reservosome complex, a site of active endocytosis and vesicle fusion present only in the epimastigote stage. Therefore, TcRab11 may represent the first molecular marker of this peculiar organelle.

Amino Acid Sequence↗
PubMed2000

The fine structure of Garnia gonadati and its association with the host cell.

Most of the studies on the fine structure of protozoa of the Apicomplexa group have been carried out with members of the ToxoPlasma, Eimeria, and Plasmodium genera. In the present study we analyzed the fine structure of Garnia gonadoti parasitizing the red blood cells of the Amazonian reptile Gonatodes humeralis (Reptilia; Lacertilia). Transmission electron microscopy of thin sections showed that G. gonadoti presented all structures characteristic of the group, including the apicoplast. However, four special features were observed: (1) absence of the hemozoin (malarial) pigment; (2) a group of microtubules associated with the mitochondrion; (3) a vacuole containing electron-dense material, which resembled the acidocalcisome described in trypanosomatids; and (4) a special array of the host-cell endoplasmic reticulum around the parasitophorous vacuole.

Animals↗
PubMed2000

Localization of saponin-sterol complexes and lectin-binding sites during interaction of Toxoplasma gondii with host cells.

The attachment of tachyzoites of Toxoplasma gondii to the surface of host cells induces the formation of a vacuole known as the parasitophorous vacuole (PV). In the present study we used lectins labeled with either fluorescein or colloidal gold particles to follow the fate of lectin-binding sites on the host-cell surface during the process of parasite/host-cell interaction. The results obtained indicate that few, if any, lectin-binding sites are internalized together with the tachyzoites to make part of the PV membrane. However, these sites are internalized and observed in small vesicles that are seen close to the PV. We also used the freeze-fracture technique in cells treated with saponin, which reveals cholesterol-containing sites as easily identifiable elongated sulculi and depressions. Intramembranous particles, which represent membrane integral proteins, were seen in the membrane lining the PV. However, saponin-cholesterol complexes were not observed in the membrane lining the PV, although they were seen in the parasite membranes. These observations, in association with data from previous studies, show that some, but not all, membrane components of the host cell contribute to the formation of the PV membrane.

Animals↗
PubMed2000

The endocytic pathway in Entamoeba histolytica.

We studied the endocytic pathway of Entamoeba histolytica using (a) confocal laser scanning microscopy to observe living cells labeled with fluorescent probes and (b) transmission electron microscopy of cells incubated in the presence of gold-labeled proteins or in a cytochemical medium designed for the localization of acid phosphatase activity. Images of acridine-orange-labeled cells showed that most of the intracellular vacuoles were acidic and were also labeled with Lucifer yellow, a fluorescent dye widely used for labeling of compartments of the endocytic pathway. A similar labeling pattern was observed when the cells were incubated in the present of fluorescein-labeled bovine albumin. However, no labeling was observed when fluorescein-labeled transferrin was used. Gold-labeled proteins (albumin, transferrin, horseradish peroxidase, and lactoferrin) were used for further characterization of the endocytic pathway. With the exception of transferrin, all the proteins bound to the protozoan surface and were subsequently internalized, appearing in small peripheral vesicles and some tubular structures. The ingested molecules accumulated in large vesicles located in the more central portion of the cell, which also presented acid phosphatase activity.

Animals↗
PubMed2000

Redistribution of plasma-membrane surface molecules during formation of the Leishmania amazonensis-containing parasitophorous vacuole.

Leishmania amazonensis presents two developmental stages that gain access to the host macrophage through phagocytosis. The protozoan resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which can fuse with the endocytic system. For evaluation of the parasite/host-cell interaction process and of PV biogenesis, the two parasite forms or host-cell membrane whose surface had previously been labeled with specific probes for lipids, proteins, and sialoglycoconjugates were allowed to interact for periods varying from 5 to 15 min for adhesion and from 30 to 60 min for PV formation. The fate of fluorescent probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, DTAF and FITC-thiosemicarbazide, which label membrane lipids, proteins, and sialoglycoconjugates, respectively, interaction with both protozoan forms revealed that adhesion to the macrophage was sufficient for labeling of the parasite surface. In addition, recently formed PVs displayed strongly labeled intravacuolar parasites, except for amastigote-macrophage interaction in a DTAF-labeled macrophage that displayed slight labeling of intravacuolar parasites, with the membrane lining the PV evidently being stained. Therefore, the vacuole modulation presents some particularities such that different host-cell membrane components may be selected, depending on the protozoan form involved. Thereafter, amastigotes labeled with the probes mentioned above displayed a diffuse labeling pattern after interaction with unlabeled macrophages, suggesting the spreading of Leishmania surface molecules during the initial parasite-invasion stages. In particular, intravacuolar DTAF-labeled amastigotes showed a delineating halo around the PV, with the intravacuolar parasite being partially labeled. Promastigotes could not be labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) or with fluorescein-5-thiosemicarbazide, but promastigotes labeled with PKH26 lost the fluorescent probe during the invasion process such that slightly labeled promastigotes were seen inside the PV. These observations indicate the existence of a dynamic process of exchange of membrane-associated glycoproteins and lipids between the parasite and the host cell.

Animals↗
PubMed2000

Nitric oxide is not involved in the killing of Trypanosoma cruzi by chicken macrophages.

It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-gamma and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii.

Animals↗
PubMed2000

The fine structure of acidocalcisomes in Trypanosoma cruzi.

Trypanosoma cruzi survives in vertebrate and invertebrate hosts and has developed mechanisms that allow it to adapt to changes in the microenvironment such as temperature, pH, and ionic composition. Most of its calcium is concentrated in an organelle named the acidocalcisome, which is acidified by a (V-H+)-adenosine triphosphatase and has H+/Ca2+ counter-transportation for calcium uptake. In this work, acidocalcisomes were examined using different transmission electron microscopy techniques. In thin sections of different stages, acidocalcisomes presented a circular shape with an electron-dense inclusion containing P3-, Ca2+, Na+, Mg2+, K+, and Zn2+. They could be distinguished from gold-labeled albumin-containing reservosomes in whole epimastigotes, and a morphometric analysis showed higher amounts of these organelles in amastigotes as compared with epimastigotes and trypomastigotes. It is possible that this variation in the amount of acidocalcisomes in the different evolutive stages could reflect adaptation mechanisms used by the parasite to survive and multiply in different environmental conditions.

Animals↗
PubMed2000

Biochemical and ultrastructural changes in Herpetomonas roitmani related to the energy metabolism.

Herpetomonas roitmani, a non-pathogenic trypanosomatid was grown in chemically defined media either containing proline or glucose as carbon source. Using transmission electron microscopy we observed that cells grown in the presence of proline present more lipid inclusions, and a larger mitochondrion with more cristae and higher activity of succinate cytochrome c reductase. On the other hand, cells grown with glucose as carbon source had more glycosomes, which were preferentially located close to the bacterium endosymbiont, and a much higher activity of hexokinase, a typical glycosome marker. Three-dimensional reconstruction and morphometrical analysis confirm these observations. The number of promastigotes of H. roitmani increased in the presence of proline. Taken together these results indicate that the growth conditions markedly influenced the ultrastructure and the metabolism of H. roitmani.

Animals↗
PubMed2000

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus.

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

Animals↗
PubMed2000

Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth.

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Antibodies, Fungal↗
PubMed2000

Two special organelles found in Trypanosoma cruzi.

We review here two unique organelles from Trypanosoma cruzi. One of them is the acidocalcisome, cytoplasmic vacuoles containing a very high Ca2+ concentration and a Ca2+ - H+ translocating ATPase activity, present in all trypanosomatids. The other organelle is the reservosome, site of accumulation of endocytosed macromolecules, very rich in cysteine proteinase, that is present only in epimastigote forms of trypanosomes belonging to the Schyzotrypanum sub-genus.

Animals↗