99m-Technetium binding site in bone marrow mononuclear cells.

INTRODUCTION: The increasing interest in 99m-technetium ((99m)Tc)-labeled stem cells encouraged us to study the (99m)Tc binding sites in stem cell compartments. METHODS: Bone marrow mononuclear cells were collected from femurs and tibia of rats. Cells were labeled with (99m)Tc by a direct method, in which reduced molecules react with (99m)Tc with the use of chelating agents, and lysed carefully in an ultrasonic apparatus. The organelles were separated by means of differential centrifugation. At the end of this procedure, supernatants and pellets were counted, and the percentages of radioactivity (in megabecquerels) bound to the different cellular fractions were determined. Percentages were calculated by dividing the radioactivity in each fraction by total radioactivity in the sample. The pellets were separated and characterized by their morphology on electron microscopy. RESULTS: The labeling procedure did not affect viability of bone marrow mononuclear cells. Radioactivity distributions in bone marrow mononuclear cell organelles, obtained in five independent experiments, were approximately 38.5 % in the nuclei-rich fraction, 5.3 % in the mitochondria-rich fraction, 2.2 % in microsomes, and 54 % in the cytosol. Our results showed that most of the radioactivity remained in the cytosol; therefore, this is an intracellular labeling procedure that has ribosomes unbound to membrane and soluble molecules as targets. However, approximately 39 % of the radioactivity remained bound to the nuclei-rich fraction. To confirm that cell disruption and organelle separation were efficient, transmission electron microscopy assays of all pellets were performed. CONCLUSIONS: Our results showed that most of the radioactivity was present in the cytosol fraction. More studies to elucidate the mechanisms involved in the cellular uptake of (99m)Tc in bone marrow cells are ongoing.

Assembly of the Leishmania amazonensis flagellum during cell differentiation.

The flagellar cytoskeleton of Leishmania promastigotes contains the canonical 9+2 microtubular axoneme and a filamentous structure, the paraflagellar rod (PFR), which is present alongside the axoneme. In contrast to promastigotes, which contain a long and motile flagellum, the amastigote form of Leishmania displays a short flagellum without a PFR that is limited to the flagellar pocket domain. Here, we investigated the biogenesis of the Leishmania flagellum at 0, 4, 6 and 24h of differentiation. Light and electron microscopy observations of the early stages of L. amazonensis differentiation showed that the intermediate forms presented a short and wider flagellum that did not contain a PFR and presented reduced motion. 3D-reconstruction analysis of electron tomograms revealed the presence of vesicles and electron-dense aggregates at the tip of the short flagellum. In the course of differentiation, cells were able to adhere and proliferate with a doubling time of about 6h. The new flagellum emerged from the flagellar pocket around 4h after initiation of cell cycle. Close contact between the flagellar membrane and the flagellar pocket membrane was evident in the intermediate forms. At a later stage of differentiation, intermediate cells exhibited a longer flagellum (shorter than in promastigotes) that contained a PFR and electron dense aggregates in the flagellar matrix. In some cells, PFR profiles were observed inside the flagellar pocket. Taken together, these data contribute to the understanding of flagellum biogenesis and organisation during L. amazonensis differentiation.

Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis.

BACKGROUND: The transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation. METHODS: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression. RESULTS: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. In intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region. CONCLUSIONS: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.

All Trypanosoma cruzi developmental forms present lysosome-related organelles.

Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidified by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have different size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo.

Biochemical properties of the major proteins from Rhodnius prolixus eggshell.

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.

Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM).

The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen's topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.

Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity.

RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.

Characterization of a RAB5 homologue in Trypanosoma cruzi.

RAB proteins are small GTPases involved in exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. RABs show a remarkable specificity in subcellular localization, so they can be used as molecular markers for studying protein trafficking in Trypanosoma cruzi, the causal agent of Chagas' disease. RAB5 is a component of early endosomes. It has been identified in kinetoplastids such as Trypanosoma brucei and Leishmania donovani. In this work, we describe the characterization of the complete coding sequence of a RAB5 gene homologue in T. cruzi (TcRAB5, GenBank Accession No. AY730667). It is present as a single copy gene, located at chromosomal bands XIII and XIV. TcRAB5 shares the highest degrees of similarity (71%) and identity (63%) with Trypanosoma brucei rhodesiense RAB5a and contains all five characteristic RAB motifs. TcRAB5 is transcribed as a single 1.5kb mRNA in epimastigotes. Its transcript was also detected in the other two forms of the parasite, metacyclic trypomastigotes and spheromastigotes. The recombinant TcRAB5 protein was able to bind and hydrolyze GTP. The identification of proteins involved in T. cruzi endo- and exocytic pathways may generate cellular compartment markers, an invaluable tool to better understand the vesicular transport in this parasite.

Cell fractionation of parasitic protozoa: a review.

Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of investigators. Here we present and discuss the work of several groups that have obtained highly purified subcellular fractions from trypanosomatids, Apicomplexa and trichomonads, and whose work have added substantially to our knowledge of the cell biology of these parasites.

Cell fractionation of parasitic protozoa: a review

Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of investigators. Here we present and discuss the work of several groups that have obtained highly purified subcellular fractions from trypanosomatids, Apicomplexa and trichomonads, and whose work have added substantially to our knowledge of the cell biology of these parasites

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70 per cent lower in the denervated extracts.