Biogenesis of the reservosomes of Trypanosoma cruzi.

Reservosomes are endocytic compartments found in the posterior region of epimastigotes of Trypanosoma cruzi. In the differentiation from trypomastigotes to epimastigotes (reverse metacyclogenesis in vitro), one has the rare opportunity of following the biogenesis of an endocytic compartment. Metacyclic trypomastigotes incubated in LIT medium highly enriched with fetal calf serum differentiated directly to epimastigotes. In recently differentiated epimastigotes, acidic organelles were found in round compartments spread along the cell body, whereas in control epimastigotes they were found in reservosomes located in the posterior region. Ultrastructural analysis of intermediate forms showed that the cytostome and reservosomes appeared before differentiation to epimastigotes was completed. Many polymorphic reservosomes, with or without lipid inclusions, were observed from the anterior portion of the cell body, in close relationship with the Golgi complex, to the posterior region. Endocytic tracers were observed in the cytostome, flagellar pocket, vesicles, and newly formed reservosomes. Cruzipain, the main protease of T. cruzi, was localized in newly formed reservosomes and in vesicles budding from the trans-Golgi network that seem to fuse with reservosomes. Ingested gold-labeled albumin and cruzipain colocalized in recently formed reservosomes. Endocytosis and immunocytochemical analysis suggested that the endocytic and the secretory pathways may contribute to reservosome formation.     

Proteomic analysis of metacyclic trypomastigotes undergoing Trypanosoma cruzi metacyclogenesis

Trypanosoma cruzi, the causative agent of the Chagas disease, has a complex life cycle alternating between replicative and noninfective forms with nonreplicative and infective forms of the parasite. Metacyclogenesis is a process that takes place in the invertebrate host, comprising morphogenetic transformation from a noninfective form to an infective form, such that parasites acquire the ability to invade human cells. We analyze here the metacyclogenesis process by 2D electrophoresis coupled to MALDI-TOF MS. A large proportion of unique proteins expressed during metacyclogenesis were observed. Interestingly, 50% of the spots were found to differ between epimastigotes and trypomastigotes. We provide a 2D map of the infective metacyclic trypomastigotes. Sixty six protein spots were successfully identified corresponding to 43 different proteins. We analyzed the expression profiles for the identified proteins along metacyclogenesis and classified them into three groups according to their maximal level of expression. We detected several isoforms for a number of proteins, some displaying differential expression during metacyclogenesis. These results suggest that posttranslational modifications may be a fundamental part of the parasite's strategy for regulating gene expression during differentiation. This study contributes to the identification of relevant proteins involved in the metacyclogenesis process. The identification and molecular characterization of these proteins will render vital information about the steps of the parasite differentiation into the infective form.     

Transition-state analysis of Trypanosoma cruzi uridine phosphorylase-catalyzed arsenolysis of uridine

Uridine phosphorylase catalyzes the reversible phosphorolysis of uridine and 2'-deoxyuridine to generate uracil and (2-deoxy)ribose 1-phosphate, an important step in the pyrimidine salvage pathway. The coding sequence annotated as a putative nucleoside phosphorylase in the Trypanosoma cruzi genome was overexpressed in Escherichia coli , purified to homogeneity, and shown to be a homodimeric uridine phosphorylase, with similar specificity for uridine and 2'-deoxyuridine and undetectable activity toward thymidine and purine nucleosides. Competitive kinetic isotope effects (KIEs) were measured and corrected for a forward commitment factor using arsenate as the nucleophile. The intrinsic KIEs are: 1'-(14)C = 1.103, 1,3-(15)N(2) = 1.034, 3-(15)N = 1.004, 1-(15)N = 1.030, 1'-(3)H = 1.132, 2'-(2)H = 1.086, and 5'-(3)H(2) = 1.041 for this reaction. Density functional theory was employed to quantitatively interpret the KIEs in terms of transition-state structure and geometry. Matching of experimental KIEs to proposed transition-state structures suggests an almost synchronous, S(N)2-like transition-state model, in which the ribosyl moiety possesses significant bond order to both nucleophile and leaving groups. Natural bond orbital analysis allowed a comparison of the charge distribution pattern between the ground-state and the transition-state models.     

Synthesis of the Trypanosoma cruzi LPPG heptasaccharyl myo-inositol

Synthesis of the heptasaccharyl myo-inositol found in Trypanosoma cruzi lipopeptidophosphoglycan was accomplished using a convergent assembly of three building blocks. The target compound is the first complete 2-aminoethyl phosphonic acid substituted glycan related to the glycosylphosphatidylinositol anchor family to be synthesized. The order of assembly enables synthesis of phosphoinositol oligosaccharides related to other glycosylinositolphospholipids in Tr. cruzi, the protozoan parasite causing Chagas' disease, which is endemic in South America.     

Epitaxial morphologies of polyoxymethylene. I. Electron microscopy

Epitaxial crystallization of polyoxymethylene (POM) from 0.5% iodobenzene solution has been attempted between 150 and 165°C on 11 different substrates having surface energies ranging from 136 to 1240 ergs/cm². Included in this series are several substrates such as CaF₂, SrF₂, BaF₂, graphite, mica, and silicon which had not been previously tested as substrates for POM epitaxy. The fluorite series materials are not good substrates for the epitaxial crystallization of POM from solution. In contrast, both rodlike and fold-plane epitaxial morphologies have been found on the (001) surfaces of NaCl, NaF, and mica. A possible mechanism for the formation of fold-plane epitaxy is discussed which involves solution nucleation followed by adsorption and growth of the nuclei on the substrate. It is suggested that variations observed in nucleation density of rodlike epitaxial crystals from substrate to substrate are a result of differences in degree of preferential solvent adsorption. Substrates having higher surface energies should tend to adsorb solvent more strongly and thus inhibit profuse nucleation of polymer crystals on the surface.     

Electron microscopy studies of potassium sodium niobate ceramics.

Using electron microscopy, K0.5Na0.5NbO3 (KNN) ceramics sintered at 1030 degrees C for 8 h and 1100 degrees C for 2 and 24 h was studied. The scanning electron microscopy and X-ray spectrometry revealed that the materials consisted of a matrix phase in which the (Na+K)/Nb ratio corresponded closely to the nominal composition and a small amount of Nb-rich secondary phase. A bimodal microstructure of cube-shaped grains was revealed in the fracture and thermally-etched surfaces of the KNN. In the ceramics sintered at 1100 degrees C, the larger grains (up to 30 mum across), contained angular trapped pores. The transmission electron microscopy analysis revealed that the crystal planes of the grains bordering the intragranular pore faces were of the {100} family with respect to the simple perovskite cell. Ferroelectric domains were observed in the grains of this material.     

Preparation of sialylated oligosaccharides employing recombinant trans-sialidase from Trypanosoma cruzi

Terminally sialylated oligosaccharides were synthesised employing recombinant trans-sialidase from Trypanosoma cruzi. Regio- and stereoselectively Sia-alpha(2-3)-Gal-betaR derivatives could be obtained in respectable yields, using combined chemical and enzymatic methodologies. An array of different disaccharide precursors such as Gal-beta(1-3)-GalNAc-alphaSer/Thr, lactosides and lactosamide derivatives were sialylated and successfully purified by facile isolation procedures. Depending on the acceptor structure isolated, yields for trans-sialylation products were between 20 and 60%.     

Electron microscopy of drawn polypropylene

Extremely thin polypropylene films formed by evaporation of dilute solutions floating on water, thin films deposited on Mylar or on carbon-coated Mylar, and bulk samples were deformed; after etching with aqua regia or chromic acid, the surfaces were studied by electron microscopy of surface replicas. At small draw ratio, microfibrils with lateral dimensions of about 200 Å, originating in micronecks at crack boundaries of the original crystal lamellae, were obtained in isolated areas exhibiting maximum local strain separated by large regions of much less deformed material. With increasing draw ratio the necked regions grow, the old structure gradually being reduced to smaller and smaller islands until it disappears completely. The inhomogeneity of strain in adjacent bundles of microfibrils creates a great many longitudinal voids with more or less disoriented microfibrils bridging the gaps. The regular arrangement of crystalline blocks of rather uniform length and width can be occasionally seen on surface replicas of drawn samples, and much better on dark-field electron micrographs of drawn and annealed thin membranes. In the latter case the blocks are very uniform and have similar dimensions along and perpendicular to the axis of the microfibril. The evidence from the electron micrographs, together with previous small-angle x-ray scattering data, supports Peterlin's molecular model for plastic deformation of crystalline polymers.     

Electron microscopy study of exotic nanostructures of cadmium sulfide.

In this article, two simple methods, evaporation-condensation and catalytic thermal evaporation, were used to investigate the synthesis of CdS nanostructures for nanoscale optoelectronic applications. To understand their growth mechanisms, various electron microscopy and microanalysis techniques were utilized in characterizing their morphologies, internal structures, growth directions and elemental compositions. The electron microscopy study reveals that when using the evaporation-condensation method, branched CdS nanorods and self-assembled arrays of CdS nanorods were synthesized at 800 degrees C and 1000 degrees C, respectively. Instead of morphological differences, both types of CdS nanorods grew along the [0001] direction. However, when using the catalytic thermal evaporation method (Au as the catalyst), patterned CdS nanowires and nanobelts were formed at the temperature region of 500-600 degrees C and 600-750 degrees C, respectively. Their growth direction was along the direction [1010] instead of [0001]. Based on the microscopy and microanalysis results, we propose some growth mechanisms in relation to the growth processes of those exotic CdS nanostructures.     

Intrinsic and Chemotherapeutic Stressors Modulate ABCC-Like Transport in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6–7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.     

Selective inhibition of Trypanosoma cruzi GAPDH by "bi-substrate" analogues

A new series of "bi-substrate" analogues have been synthesized as potential inhibitors of the glyceraldehyde-3-phosphate dehydrogenase and one lead compound has been identified that inhibits the enzyme from Trypanosoma cruzi with good affinity and very high (50-fold) specificity.     

Electron microscopy observations on the role of twinning in the evolution of microstructures.

Twinning plays an important role in phase transformations and can have significant effects on microstructural evolution. Different roles of twinning in the development of microstructures during precipitation and phase transformations are reviewed and illustrated with examples from investigations by high-resolution electron microscopy, including the effect of multiple twinning on the development of Ge precipitates in Al-Ge and Ag-Ge alloys, the twin dissociation of grain boundaries in Au, the formation of hexagonal Si at twin intersections and the effect of twin boundaries on the equilibrium shape of Pb inclusions in Al.     

In vitro activity of compounds isolated from Piper crassinervium against Trypanosoma cruzi

This study describes the antichagasic potential of five compounds isolated from leaves of Piper crassinervium (Piperaceae). Two prenylated benzoic acid derivatives, one prenylated hydroquinone and two flavanones, were evaluated. The in vitro trypanocidal activity was determined against epimastigote forms of Trypanosoma cruzi (Y strain), the etiologic agent of Chagas disease. The most active compound was the prenylated hydroquinone [1,4-dihydroxy-2-(3(0),7(0)-dimethyl-1(0)-oxo-2(0)-E,6(0)-octadienyl)benzene] with an IC(50) value of 6.10 microg mL(-1), which was in the same order of activity if compared with the positive control benznidazole (IC(50) = 1.60 microg mL(-1)). This is the first report of trypanocidal activity for prenylated hydroquinone and benzoic acid derivatives.     

Electron microscopy investigations of the morphology of a polyester-polyurethane

In the current paper the morphology of a slightly crosslinked polyester-polyurethane (hard-segment content ca. 33 wt.-%) is studied by means of transmission electron microscopy (TEM). A new staining technique based on ruthenium tetroxide was applied. This new technique allows the unambiguous visualization of the typical microdomain structure of the polyurethane and also of a wide size spectrum of globular hard-segment-rich domains which result from phase separation in the early stage of preparation. The hard-segment-rich microdomains have a size between 1.5 and 10 nm while the globular domains span a size spectrum from 10 to 2000 nm.     

Cytotoxicity of Essential Oil Cordia verbenaceae against Leishmania brasiliensis and Trypanosoma cruzi

The species Cordia verbenacea DC (Boraginaceae), known as the whaling herb and camaradinha, is a perennial shrub species native to the Atlantic Forest. Its leaves are used in folk medicine as an anti-inflammatory, analgesic, antiulcerogenic and curative agent, in the form of teas or infusions for internal or topical use. The present study aimed to verify the cytotoxicity of the essential oil and the leishmanicidal and trypanocidal potential of C. verbenacea. The essential oil was characterized by GC-MS. The in vitro biological activity was determined by anti-Leishmania and anti-Trypanosoma assays. The cytotoxixity was determined using mammalian fibroblasts. The C. verbenacea species presented α-pinene (45.71%), β-caryophyllene (18.77%), tricyclo[2,2,1-(2.6)]heptane (12.56%) as their main compounds. The essential oil exhibited strong cytotoxicity at concentrations below 250 μg/mL (LC₅₀ 138.1 μg/mL) in mammalian fibroblasts. The potent anti-trypanosome and anti-promastigote activities occurred from the concentration of 62.5 μg/mL and was considered clinically relevant. The results also demonstrate that at low concentrations (<62.5 μg/mL), the essential oil of C. verbenacea managed to be lethal for these activities. This can be considered an indication of the power used in daily human consumption. Therefore, it can be concluded that the essential oil of C. verbenacea contains a compound with remarkable antiparasitic activities and requires further research.     

Bisphosphonate metal complexes as selective inhibitors of Trypanosoma cruzi farnesyl diphosphate synthase

In the search for a pharmacological answer to treat Chagas disease, eight metal complexes with two bioactive bisphosphonates, alendronate (Ale) and pamidronate (Pam), were described. Complexes of the formula [M(2)(II)(Ale)(4)(H(2)O)(2)]·2H(2)O, with M = Cu, Co, Mn, Ni, and ([CuPam]·H(2)O)(n) as well as [M(II)(Pam)(2)(H(2)O)(2)]·3H(2)O, with M = Co, Mn and Ni, were synthesized and fully characterized. Crystal structure of [Cu(2)(II)(Ale)(4)(H(2)O)(2)]·2H(2)O, [Co(II)(Pam)(2)(H(2)O)(2)] and [Ni(II)(Pam)(2)(H(2)O)(2)] were solved by X-ray single crystal diffraction methods and the structures of [M(2)(II)(Ale)(4)(H(2)O)(2)]·2H(2)O complexes M = Co, Mn and Ni were studied by X-ray powder diffraction methods. All obtained complexes were active against the amastigote form of Trypanosoma cruzi (T. cruzi), etiological agent of Chagas disease. Most of them were more active than the corresponding free ligands showing no toxicity for mammalian cells. The main mechanism of the antiparasitic action of bisphosphonates, inhibition of parasitic farnesyl diphosphate synthase (TcFPPS), remains in the obtained metal complexes and an increase in the inhibiting enzyme levels was observed upon coordination. Observed enzymatic inhibition was selective for TcFPPS as the metal complexes showed no or little inhibition of human FPPS. Additionally, metal complexation might improve the bioavailability of the complexes through the hindrance of the phosphonate group's ionization at physiological pH and, eventually, through the ability of plasma proteins to work as complex transporters.     

Electron microscopy and quantitative polymer morphology

The lamellar nature of semi-crystalline polymers can now be studied systematically using transmission electron microscopy, especially following permanganic etching. In combination with thermal analysis and selective extraction followed by molecular characterisation, this approach allows the distribution of lamellae, thermal stability and molecular constitution to be mapped in real space. Extension of this methodology has allowed the extraction of individual melt-crystallized lamellae from bulk polymer. Their study is adding to understanding of fundamental processes of crystal growth.