Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures

Characterization Of The Intracellular Ca2+ Pools Involved In The Calcium Homeostasis In Herpetomonas Sp. Promastigotes

Trypanosomatids of the genus Herpetomonas comprises monoxenic parasites of insects that present proand opisthomastigotes forms in their life cycles. In this study, we investigated the Ca2+ transport and the mitochondrial bioenergetic of digitonin-permeabilized Herpetomonas sp. promastigotes. The response of promastigotes mitochondrial membrane potential to ADP, oligomycin, Ca2+, and antimycin A indicates that these mitochondria behave similarly to vertebrate and Trypanosoma cruzi mitochondria regarding the properties of their electrochemical proton gradient. Ca2+ transport by permeabilized cells appears to be performed mainly by the mitochondria. Unlike T. cruzi, it was not possible to observe Ca2+ release from Herpetomonas sp. mitochondria, probably due to the simultaneous Ca2+ uptake by the endoplasmic reticulum. In addition, a vanadate-sensitive Ca2+ transport system, attributed to the endoplasmic reticulum, was also detected. Nigericin (1 μM), FCCP (1 μM), or bafilomycin A1 (5 μM) had no effect on the vanadate-sensitive Ca2+ transport. These data suggest the absence of a Ca2+ transport mediated by a Ca2+/H+ antiport. No evidence of a third Ca2+ compartment with the characteristics of the acidocalcisomes described by A. E. Vercesi et al. (1994, Biochem. J. 304, 227-233) was observed. Thapsigargin and IP3 were not able to affect the vanadate-sensitive Ca2+ transport. Ruthenium red was able to inhibit the Ca2+ uniport of mitochondria, inducing a slow mitochondrial Ca2+ efflux, compatible with the presence of a Ca2+/H+ antiport. Moreover, this efflux was not stimulated by the addition of NaCl, which suggests the absence of a Ca2+/Na+ antiport in mitochondria. (C) 2000 Academic Press.38018591Camargo, E.P., Kastelein, P., Roitman, I., (1990) Parasitol. Today, 6, pp. 22-25Redman, C.A., Schineider, P., Mehlert, A., Ferguson, A.J., (1995) Biochem. 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Volutin Granules of Eimeria Parasites are Acidic Compartments and Have Physiological and Structural Characteristics Similar to Acidocalcisomes

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.585416423Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)US National Institutes of Health [AI-07965]Barbara and Sanford Orkin/Georgia Research Alliance ChairConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)US National Institutes of Health [AI-07965

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional Conselho Nacional de Pesquisa e Desenvolvimento Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Volutin Granules Of Eimeria Parasites Are Acidic Compartments And Have Physiological And Structural Characteristics Similar To Acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites. © 2011 International Society of Protistologists.585416423Bakar, N.A., Klonis, N., Hanssen, E., Chan, C., Tilley, L., Digestive vacuole genesis and endocytic processes in the early intraerythrocytic stages of Plasmodium falciparum (2009) J. Cell. 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Parasitenk., 28, pp. 115-121De Souza, W., Special organelles of some pathogenic protozoa (2002) Parasitology Research, 88 (12), pp. 1013-1025. , DOI 10.1007/s00436-002-0696-2Docampo, R., Special section on novel organelles in parasitic protozoa (2008) Experimental Parasitology, 118 (1), p. 1. , DOI 10.1016/j.exppara.2007.12.005, PII S0014489407003165Docampo, R., Moreno, S.N., Bisphosphonates as chemotherapeutic agents against trypanosomatid and apicomplexan parasites (2001) Curr. Drug Targets Infect. Disord., 1, pp. 51-61Docampo, R., Moreno, S.N.J., The acidocalcisome as a target for chemotherapeutic agents in protozoan parasites (2008) Current Pharmaceutical Design, 14 (9), pp. 882-888. , http://www.ingentaconnect.com/content/ben/cpd/2008/00000014/00000009/ art00007, DOI 10.2174/138161208784041079Docampo, R., De Souza, W., Miranda, K.R., Rohloff, P., Moreno, S.N.J., The acidocalcisome-conserved from bacteria to man (2005) Nat. Rev. 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Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae)

Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K-0.5 values for Na+ with minor alterations in K-0.5 values for K+ and N-H-4(+), causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 mu M ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. (c) 2008 Elsevier Inc. All rights reserved