Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

In vitro and in vivo activities of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol against Trypanosoma cruzi.

From a series of 1,3,4-thiadiazole-2-arylhydrazone derivatives of megazol screened in vitro against Trypanosoma cruzi, eight (S1 to S8) were selected for in vivo screening by single-dose oral administration (200 mg/kg of body weight) to infected mice at 5 days postinfection (dpi). Based on significant decreases in both parasitemia levels and mortality rates, S2 and S3 were selected for further assays. Despite having no in vivo effect, S1 was included since it was 2-fold more potent against trypomastigotes than megazol in vitro. Trypomastigotes treated with S1, S2, or S3 showed alterations of the flagellar structure and of the nuclear envelope. When assayed on intracellular amastigotes, the selectivity index (SI) for macrophages was in the range of >27 to >63 and for cardiac cells was >32 for S1 and >48 for megazol. In noninfected mice, S1 did not alter the levels of glutamic oxalacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), or urea. S2 led to an increase in GOT, S3 to increases in GOT and GPT, and megazol to an increase in GOT. Infected mice were treated with each derivative at 50 and 100 mg/kg from dpi 6 to 15: S1 did not interfere with the course of infection or reduce the number of inflammatory foci in the cardiac tissue, S2 led to a significant decrease of parasitemia, and S3 decreased mortality. There was no direct correlation between the in vitro effect on trypomastigotes and amastigotes and the results of the treatment in experimental models, as S1 showed a high potency in vitro while, in two different schemes of in vivo treatment, no decrease of parasitemia or mortality was observed.

Image analysis of two-dimensional gel electrophoresis for comparative proteomics of transgenic and non-transgenic soybean seeds.

Considering the importance of bidimensional electrophoresis and image analysis in comparative proteomics, the parameters that influence the analysis of protein expression of transgenic and non-transgenic soybean seeds were evaluated. The loaded mass of the proteins (150-500 microg), the pH separation range (3-10 or 4-7), and manual/automatic image editing were evaluated. Additionally, after optimizing the conditions, histograms and matchings were obtained in order to accurately analyze the variations (90%) in protein expression. From this, 10 proteins displayed significant differences in expression, and eight of them were characterized and identified by mass spectrometry.

Dual affinity method for plasmid DNA purification in aqueous two-phase systems.

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-gamma levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-beta was high, whereas IL-12 and TNF-alpha were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.

Ultrastructural analysis of edelfosine-treated trypomastigotes and amastigotes of Trypanosoma cruzi.

We have previously reported that epimastigote forms of Trypanosoma cruzi treated with the lysophospholipid analogues (LPAs) edelfosine (ET-18), ilmofosine, and miltefosine suffered alterations in plasma membrane, mitochondrion, and lipid synthesis. In this work, ET-18 induced membrane damage in trypomastigotes and amastigotes. Incubation of epimastigotes and trypomastigotes with the three LPAs led to membrane permeabilization, which was abolished by serum addition. Treatment for 24 h in culture medium interfered the with mitochondrial status of epimastigotes, with no effect in trypomastigotes, in agreement with ultrastructural data. LPAs induced alterations in the plasma membrane of the three forms of T. cruzi and in the mitochondria of epimastigotes, suggesting that these organelles are potential targets of these analogues.

Absence of vacuolar membrane involving Toxoplasma gondii during its intranuclear localization.

Tachyzoites of Toxoplasma gondii were located inside the nucleus of both skeletal muscle cells infected in vitro and peritoneal exudate cells collected from infected mouse in vivo. Ultrastructural analysis demonstrated that T. gondii invades the nucleus of host cells by the parasite apical region and with constriction of its body. We noted that the rhoptry, a secretory organelle of the parasite that is involved in the host cell invasion mechanism, was empty in the intranuclear T. gondii. The parasites were found in the nuclear matrix without evidence of the vacuolar membrane. Frequently, new parasites invaded host cell nucleus, which was already infected. The significance of this nuclear invasion could reflect an alternative route of T. gondii for its transitory survival or an escape mechanism from the host immune response during the in vivo infection (or both).

Redescription and surface ultrastructure of Pygidiopsis macrostomum (Digenea: Heterophyidae).

Pygidiopsis macrostomum Travassos, 1928, a poorly known species originally described from a single specimen from Rattus norvegicus (Erxleben, 1777) in Brazil, is redescribed on the basis of metacercariae from the mesenteries of naturally infected guppies Poecilia vivipara Bloch and Schneider, 1801 (Poeciliidae), and adults obtained from an experimental infection of hamsters. Pygidiopsis macrostomum is characterized by the absence of oral spines, vitellaria extending forward to ventral sucker, uterus reaching pharyngeal level, X-shaped excretory vesicle, and an oral sucker/acetabulum ratio of 1:0.8. The surface ultrastructure shows that the tegument of the metacercaria does not strongly differ from that of adults. The brush-shaped spines of P. macrostomum are similar to those reported for Pygidiopsis summa and Pygidiopsis ardeae, but no differences in spine shape were observed throughout the body.

Biological and ultrastructural effects of the anti-microtubule agent taxol against Trypanosoma cruzi.

Microtubules play fundamental roles in eukaryotic cells and have been investigated as target for drugs. Several studies showed the potential use of anti-microtubule agents against pathogenic protozoa. Taxol has been intensively studied in Leishmania spp. and microtubules have been considered as a promising antileishmanial drug target. It has been also shown that taxol interferes with the proliferation of Trypanosoma cruzi, leading to morphological alterations and interruption of nuclear division and cytokinesis. In the present work we show that T. cruzi bloodstream trypomastigotes were much more susceptible than epimastigotes, and in both forms taxol caused severe ultrastructural damage, especially associated to changes in the shape of the parasites. In trypomastigotes, different degrees of body contortion along the longitudinal axis and a marked dilatation of the flagellar pocket were detected. Treated epimastigotes presented a decrease in the electron density of the mitochondrial matrix, absence of mitochondrial cristae and an increase in the number of lipid droplets. Bizarre multi-flagellar epimastigotes were also detected, suggesting an interruption of the cytokinesis. Taxol caused no noticeable ultrastructural alterations on sub-pellicular and flagellar microtubules of both evolutive forms of T. cruzi. As already described in the literature, such structures in trypanosomatids are very resistant to microtubule disrupters when compared to those in mammalian cells. Taxol prevented the endocytosis of albumin-gold complexes by epimastigotes, and this result could be associated to the loss of the dynamic stability of the microtubules of the cytostome.

Comparative ultrastructural analysis of the antennae of Triatoma guazu and Triatoma jurbergi (Hemiptera: Reduviidae) during the nymphal stage development.

The specific concept of two triatominae species of epidemiological importance in the Mato Grosso Region (Brasil), Triatoma guazu Lent & Wygodzinsky, 1979 and Triatoma jurbergi Carcavallo, Galvão & Lent, 1998, the antenniferous tubercle and the four antennal segments of nymphs from the first to fifth instar were morphologically compared by scanning electron microscopy. The main differences observed were that the antenniferous tubercle in T. guazu did not present a smaller tubercle in the base of the larger tubercle. The first antennal segment in the fifth instar had sensilla distributed with an alternating array and the trichobothria in the first instar had half of its length reaching the third antennal segment. However, in T. jurbergi the antenniferous tubercle had two smaller tubercles in the base of the two larger tubercles. The first antennal segment in the fifth instar presents sensilla distributed in pairs, and the trichobothria in the first instar has only a small portion of the structure reaching the third antennal segment. These structures differentiated the nymphs of T. guazu and T. jurbergi.

Dinitroaniline herbicides against protozoan parasites: the case of Trypanosoma cruzi.

The drugs presently in use against Chagas disease are very toxic, inducing a great number of side effects. Alternative treatments are necessary, not only for Chagas disease but also for other diseases caused by protozoan parasites where current drugs pose toxicity problems. The plant microtubule inhibitor trifluralin has previously been tested with success against Leishmania, Trypanosoma brucei and several other protozoan parasites. Trypanosoma cruzi, the causative agent of Chagas disease, is also sensitive to the drug. This sensitivity has been correlated with the deduced amino acid sequences of alpha- and beta-tubulin of T. cruzi as compared with plant, mammal and other parasite sequences.

Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.

Geographic diversity of adult t-cell leukemia/lymphoma in Brazil. The Brazilian ATLL Study Group.

We describe 195 cases of adult T-cell leukemia/lymphoma (ATLL) reported to the national registry of T-cell malignancies in Brazil between 1994 and 1998. We compared the effect of demographic differences and clinical features of 150 consecutive ATLL cases in different regions of this diverse country. At diagnosis, the predominant clinical sub-type was the acute type (60%), followed by lymphoma (22%), chronic (10%) and smoldering (8%) types. Although we expected that different sub-types would be present in different regions, on the basis of immunogenetic factors determined by ethnicity, we did not demonstrate these differences. There were no significant differences among ATLL subtypes by age or gender. No ethnic group predominated in the total population of patients, but significant differences were noted when examining ethnic distribution by region. Reflecting the general population distribution, white patients were seen more often in São Paulo and black patients in Bahia, than in other regions. In most regions, cases were equally distributed between blacks and mulattos, except in Pernambuco, where blacks were less frequent. The main clinical features were lymphadenopathy, skin lesions, hypercalcemia and hepatomegaly. Fourteen patients (9%) suffered from HTLV-I-associated myelopathy (HAM/TSP), either at diagnosis or during follow-up of ATLL. All cases but one had antibodies to HTLV-I, with concordant results with ELISA, WB and PCR analyses. For the antibody-negative case, pol and tax gene sequences were present in tumor cells when subjected to PCR analyses. The prognosis was generally poor, suggesting that the disease in Brazil behaves in similar fashion regardless of ethnic or geographical differences.

A cardiomyocyte mannose receptor system is involved in Trypanosoma cruzi invasion and is down-modulated after infection.

Mannosyl binding sites were detected "in vitro" on cardiomyocytes (CM) surface using horseradish peroxidase (HRP) as the ligand. Binding assays revealed a specific recognition system, which was time- and concentration-dependent. The binding required physiological pH and was inhibited by EDTA and trypsin treatments. HRP binding was reduced by pre-incubations with low concentrations of D-mannose. Ultrastructural analysis of the endocytic process was followed using HRP coupled to colloidal gold particles (HRP-Au). The tracer was found within caveolae characterizing early steps of the receptor-mediated endocytosis. The addition of 10 mM D-mannose to the interaction medium blocked Trypanosoma cruzi uptake by CM. The labeling of CM with a subsaturating concentration of HRP-Au before their infection showed, by ultrastructural studies, that its association with trypomastigote forms occurred frequently near to HRP-gold particles that could also be seen to comprise the parasitophorous vacuole. After infection of CM with T. cruzi, a considerable reduction on HRP binding was noticed. Binding was almost completely restored by treating the infected cultures with the trypanocidal drug Nifurtimox. Our "in vitro" findings suggest that cardiomyocyte's mannose receptors localized at the sarcolemma mediates T. cruzi recognition and can be down-modulated by parasite infection.

Frequent expansion of Epstein-Barr virus (EBV) infected cells in germinal centres of tonsils from an area with a high incidence of EBV-associated lymphoma.

Burkitt's lymphoma (BL) and Hodgkin's disease (HD) occurring in developing regions are frequently associated with Epstein-Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV-infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV-encoded small nuclear RNA (EBER) and EBV-encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER-positive lymphoid cells were found in the GCs of 8/20 EBV-positive tonsils from Brazil (9-89 cells/GC; mean: 14.5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1-9 cells/GC; mean: 0.5 cell/GC per case) in this compartment (p < 0.007). In addition, the EBV-infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV-encoded latent membrane protein (LMP)-1, findings not present in German samples. These data show that latently EBV-infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP-1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD.

Adult T-cell leukemia/lymphoma in northeastern Brazil: a clinical, histopathologic, and molecular study.

The state of Bahia in the northeastern coast of Brazil is a region in which HTLV-I infection is endemic. This study investigated the characteristics of 28 HTLV-I-associated lymphomas/leukemias in this region. HTLV-I-infection diagnosis was based on serologic study, Southern blot analysis, and polymerase chain reaction (PCR) in neoplastic tissue. The main clinical differences between these lymphomas and adult T-cell leukemia (ATL) cases from other endemic areas were as follows. The mean age was 47 years; 20% of the cases occurred in young adults; and a predominance was found among male subjects (2:1), blacks, and of those of mixed race (96%). Histologically, 20 cases were T-cell pleomorphic leukemia/lymphoma, 5 were Mycosis fungoides-like cutaneous lymphoma, and 3 were CD30+ large-cell anaplastic lymphoma. Immunohistochemistry demonstrated 4 cases of CD8+ lymphoma. Proviral genomic sequences were demonstrated by PCR in 9 lymph node biopsy specimens and in 3 skin biopsy specimens. Southern blot was performed and was positive in 8 cases.

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction.

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

Trypanosoma cruzi: resistance to the pore forming protein of cytotoxic lymphocytes--perforin.

The pore-forming protein perforin is one of the main effector molecules which cytotoxic lymphocytes utilize to kill their targets both in vivo and in vitro. Natural killer cells and cytotoxic T lymphocytes play an important role in host defense against a number of intracellular microorganisms such as virus and protozoan, but the exact way they help control infection is unknown. On the other hand, many microorganisms have evolved successful escape strategies to avoid immune-cell-mediated attack. It is thus necessary to investigate the direct interaction of infectious microorganisms with the lytic machinery of cytotoxic lymphocytes and other cells. In the present work we report the effect of perforin on both a protozoan, Trypanosoma cruzi, and the infected host cell. Epimastigote, amastigote, and trypomastigote forms of T. cruzi, as well as infected macrophages, were assayed for their susceptibility to perforin based on three different criteria. T. cruzi in all three differentiation stages were resistant to purified perforin at doses up to 100-fold larger than that sufficient to kill susceptible tumor cells. No morphological change was observed under electron microscopy. Survival rates and infectivities of the treated parasites in vitro were similar to those of control parasites. Moreover, the measurement of calcium influx using Fura-2 to assess membrane damage revealed that T. cruzi resist perforin attack by avoiding transmembrane pore formation. Resistance to perforin was not transferred to host cells since infected macrophages could be easily destroyed by perforin while intracellular amastigotes remained intact.