16 research outputs found
Venom alkaloids against Chagas disease parasite: search for effective therapies
Chagas disease is an important disease affecting millions of patients in the New World and is caused by a protozoan transmitted by haematophagous kissing bugs. It can be treated with drugs during the early acute phase; however, effective therapy against the chronic form of Chagas disease has yet to be discovered and developed. We herein tested the activity of solenopsin alkaloids extracted from two species of fire ants against the protozoan parasite Trypanosoma cruzi, the aetiologic agent of Chagas disease. Although IC50 determinations showed that solenopsins are more toxic to the parasite than benznidazole, the drug of choice for Chagas disease treatment, the ant alkaloids presented a lower selectivity index. As a result of exposure to the alkaloids, the parasites became swollen and rounded in shape, with hypertrophied contractile vacuoles and intense cytoplasmic vacuolization, possibly resulting in osmotic stress; no accumulation of multiple kinetoplasts and/or nuclei was detected. Overexpressing phosphatidylinositol 3-kinase—an enzyme essential for osmoregulation that is a known target of solenopsins in mammalian cells—did not prevent swelling and vacuolization, nor did it counteract the toxic effects of alkaloids on the parasites. Additional experimental results suggested that solenopsins induced a type of autophagic and programmed cell death in T. cruzi. Solenopsins also reduced the intracellular proliferation of T. cruzi amastigotes in infected macrophages in a concentration-dependent manner and demonstrated activity against Trypanosoma brucei rhodesiense bloodstream forms, which is another important aetiological kinetoplastid parasite. The results suggest the potential of solenopsins as novel natural drugs against neglected parasitic diseases caused by kinetoplastids.Fil: Silva, Rafael C. M. Costa. Universidade Federal do Rio de Janeiro; BrasilFil: Fox, Eduardo G. P.. Universidade Federal do Rio de Janeiro; Brasil. South China Agricultural University; ChinaFil: Gomes, Fabio M.. Universidade Federal do Rio de Janeiro; Brasil. National Institutes of Health; Estados UnidosFil: FeijĂł, Daniel F.. Universidade Federal do Rio de Janeiro; BrasilFil: Ramos, Isabela. Universidade Federal do Rio de Janeiro; BrasilFil: Koeller, Carolina M.. Universidade Federal do Rio de Janeiro; Brasil. University at Buffalo; Estados UnidosFil: Costa, Tatiana F. R.. Universidade Federal do Rio de Janeiro; BrasilFil: Rodrigues, Nathalia S.. Universidade Federal do Rio de Janeiro; BrasilFil: Lima, Ana P.. Universidade Federal do Rio de Janeiro; BrasilFil: Atella, Georgia C.. Universidade Federal do Rio de Janeiro; BrasilFil: Rocha de Miranda, Kildare. Universidade Federal do Rio de Janeiro; Brasil. Instituto Nacional de CiĂŞncia e Tecnologia em Biologia Estrutural e Bioimagem; BrasilFil: Schoijet, Alejandra Cecilia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular "Dr. HĂ©ctor N. Torres"; ArgentinaFil: Alonso, Guillermo Daniel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular "Dr. HĂ©ctor N. Torres"; ArgentinaFil: de Alcântara Machado, Ednildo. Universidade Federal do Rio de Janeiro; BrasilFil: Heise, Norton. Universidade Federal do Rio de Janeiro; Brasi
Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi
, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating
Ultrastructure of trypanosoma cruzi revisited by atomic force microscopy
7 p. : il.Most advances in atomic force microscopy (AFM) have been accomplished in recent
years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried
cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites
Acidocalcisomes in apicomplexan parasites
8 p. : il.Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic
matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their
matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase.
The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes
have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in
Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan
parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis
Calcium- and polyphosphate containing acido calcisomes in chicken egg yolk
14 p. : il., tabBackground information. Poly P (inorganic polyphosphate) is a polymer formed by Pi residues linked by high-energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis.
Results. We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4 ,6-diamidino-2-phenylindole) staining showed that poly P is localized mainly in electron-dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as
detected by X-ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (Pi) of yolk organelles, 16% is present in the form of poly P. Total
poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development.
Conclusions. The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles
with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles.
The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs
Polyphosphate polymers during early embryogenesis of Periplaneta americana
8 p. : il.Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the
embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca+2, Pi and PolyP levels were observed during the first days of
embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca+2 levels. This is the first investigation of
PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles,
might be involved in the functional control of Ca+2 and Pi homeostasis during early embryogenesis of P. Americana
Inorganic polyphosphate inhibits an aspartic protease-like activity in the eggs of rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro
6 p. : il.Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate
cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsinDis the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsinDand arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the
eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules.
Weobserved that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsinDinhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsinDinhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing
cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk
mobilization during embryogenesis of R. prolixus
Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of periplaneta americana
9 p. : il.Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of
cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase
(V-HĂľ-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the
active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In
the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in
organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded
by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is
mediated by proton-pumps present in their membranes. In this work, we describe an HĂľ-PPase activity
in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme
activity was optimum at pH around 7.0, and was dependent on Mg2Ăľ and inhibited by NaF, as well as by
IDP and Ca2Ăľ. Immunolocalization of the yolk preparation using antibodies against a conserved sequence
of V-HĂľ-PPases showed labeling of small vesicles, which also showed the presence of high concentrations
of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In
addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the
small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of
P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles
during embryogenesis of this insect is discussed
Calcium- regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl
11 p. : il.This study examined the process of membrane fusion of yolk granules (YGs) during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days·0 and 3 after oviposition contain different populations of
YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca2+] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca2+] similar to those found in day-3·eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calciumdependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H+-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery