Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites
© Cruz et al; licensee BioMed Central Ltd. 2012
Received: 5 December 2011
Accepted: 15 March 2012
Published: 15 March 2012
Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.
Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.
The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11).
The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.
KeywordsATP Purinergic receptor Malaria Plasmodium berghei Plasmodium yoelii Protease activity Calcium modulation Merozoite surface protein 1
Malaria is one of the most important infectious diseases in the world, responsible for an estimated 655,000 deaths each year . While Plasmodium grows and develops inside red blood cells (RBCs), concomitant structural  and biochemical changes occurs at the host cell culminating in cell rupture and release of free merozoites[3, 4]. It is now well established that Plasmodium activates proteases during the blood stages, including during the entry into and exit from its host RBC and the intracellular feeding on haemoglobin [5–9].
As an adaptive evolutionary mechanism, the malaria parasite subverts its host's signalling system to survive and replicate[10–12]. The role of Ca2+ signalling underlying modulation of the Plasmodium cell cycle has been extensively investigated including an effect on protease activity[13, 14]. For example, some proteases are modulated by intracellular Ca2+ in rodent Plasmodium species. Such signalling depends on the maintenance of low cytosolic Ca2+ during its RBC stages[16–26]. However, it is still unknown how a calcium signal is triggered and how particular metabolites derived from the host are central in providing signalling molecules to facilitate parasite growth. The host hormone, melatonin  and its derivatives that elicit a rise in cytosolic calcium in Plasmodium[17, 28–30] also induce proteolysis in Plasmodium falciparum and Plasmodium chabaudi.
Here we have investigated whether other metabolites derived from the host are able to induce proteolysis in Plasmodium berghei and Plasmodium yoelii. Purines such as adenosine, ADP, ATP and UDP mediate several biological processes, being essential in various metabolic cycles and extracellular signalling . The role of ATP in cell signalling is well studied in many eukaryotic cells and includes a wide variety of processes such as secretion, immune responses, mechano-sensory transduction, inflammation, platelet aggregation, cell proliferation, differentiation, and cell migration.
ATP is released from RBCs when they are deformed, and this process could also be relevant to malaria parasite invasion since RBCs undergo extensive deformation after the initial merozoite attachment. In addition, it has been reported that following Plasmodium infection, the ATP content of RBCs increases  and blocking purinergic signalling decreases RBCs invasion by P. falciparum. Interestingly, suramin, which inhibits purinergic signalling, has been shown to inhibit merozoite surface protein-1 (MSP1) processing and erythrocyte invasion. In this manner, a role of ATP in Ca2+ signalling and proteolysis to modulate the Plasmodium RBC cell cycle is hypothesized.
By using fluorescence resonance energy transfer (FRET) peptides, it was previously shown that Ca2+ modulates protease activation in P. berghei and P. yoelii parasites. Here, the importance of ATP in modulating proteolysis through Ca2+ pathways in these parasites has been investigated. In addition, the role of ATP in activating proteases or modulating the P. berghei cell cycle was studied in the presence of purinergic blocker KN-62. It is shown here that this compound blocks parasite maturation and affects processing of the merozoite surface protein MSP1. Taken together, the present work contributes to the understanding of P. berghei biology.
Materials and methods
Thapsigargin, (phenylmethylsulphonyl fluoride), saponin, probenecid, MOPS (3-(N-morpholino) propanesulfonic acid), EGTA (ethylene glycol-bis (2-aminoethylether)-N,N,N',N tetraacetic acid), adenosine, ATP (adenosine-5'-triphosphate), GTP (guanosine-5'-triphosphate), suramin, PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid), IP5I (diinosine pentaphosphate), TNP-ATP (3'-O-(2,4,6-Trinitrophenyl)adenosine-5'-triphosphate tetra(triethylammonium) salt), KN-62 (4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester), Triton X-100 and dihydroethidium were purchased from Sigma- Aldrich (St. Louis, MO). BAPTA/acetoxymethyl ester (AM) and Fluo4/AM were bought from Molecular Probes Inc. (Eugene, OR). The peptide Abz-AIKFFARQ-EDDnp was analytical grade and synthesized according to Hirata, 1994[39–41].
Plasmodium berghei (strain NK65) and P. yoelii (strain 17X) parasites
Plasmodium berghei and P. yoelii were maintained as an asynchronous parasitaemia in mice (Balb/C strain) by transfer every four days. For parasite preparation, filtration of the infected blood through a cellulose column (Whatman CF11) removed leukocytes and platelets. The erythrocytes were then washed twice in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM NaH2PO4,) by centrifugation at 1,500 g for 5 min and lysed in PBS containing 60 μg ml-1 saponin. The membranes were removed by centrifugation (10,000 × g for 10 min at 4°C) and further washing of the parasites (1,500 g for 5 min) in MOPS buffer (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM D-glucose, 50 mM MOPS, and 1 mM CaCl2, pH 7.2) containing saponin. After erythrocyte lysis, the parasites were maintained in MOPS buffer during the whole experiment.
All animal procedures were approved by the São Paulo University Ethics Committee for Animal Experiments (CEEA) according to the Colégio Brasileiro de Experimentação Animal guidelines (COBEA).
Cell culture of Plasmodium berghei (strain NK65) parasites
Plasmodium berghei parasites in mice (Balb/C) were transferred to culture at a parasitaemia of 6-10%. The infected RBCs were filtered through a cellulose column (Whatman CF11) as described above and washed twice with RPMI 1640 medium (GIBCO BRL) supplemented with 10% foetal calf serum (FCS). Infected RBCs were then transferred to a culture chamber and kept in suspension by a magnetic stirrer, under an atmosphere of 5% O2, 7% CO2 and 88% N2. The parasites were maintained in culture for 17 h. The stages of intraerythrocytic development were determined by morphology on Giemsa-stained smears.
Peptide and calcium indicator Fluo4/AM loading
The FRET peptide Abz-AIKFFARQ-EDDnp has a fluorescent group, Abz (ortho-aminobenzoic acid), and a quencher group, EDDnp (ethylene diamine-2-4-dinitrophenyl). The peptide is able to access free malaria parasites when the erythrocyte membrane was removed by saponin treatment in MOPS buffer after 1 min incubation. Stock solutions were prepared in DMSO/water (1:1) and concentrations were measured spectrophotometrically using a molar absorption coefficient of 17,300 M-1 cm-1 at 365 nm. For Ca2+ measurements isolated parasites were incubated for 30 min at room temperature with the fluorescent calcium indicator Fluo-4/AM (5 μM) in MOPS buffer containing 1.8 mM probenecid, an inhibitor of organic transport, to minimize indicator extrusion. The cell suspension was then washed three times in MOPS buffer to remove the extracellular dye.
Spectrofluorimeric measurements were performed in a Shimadzu RF-5301 PC at 37°C with isolated parasites (108 cells ml-1) incubated with MOPS buffer in a 1 ml cuvette. The fluorescence was measured continuously (acquisition rate: every 0.5 seconds) 1 min after addition of the FRET peptide (10 μM) for 400 seconds. Excitation/emission wavelengths were adjusted to 320/420 nm for Abz and 505/530 nm for Fluo-4 AM. For experiments with purinergic inhibitors parasites were pre-incubated with KN-62, IP5I, TNP-ATP, PPADS or suramin, for 30 min at room temperature. For experiments with the extra or intracellular calcium chelator EGTA (5 mM) or Bapta/AM (25, 100, 200 or 500 μM) parasites were pre-incubated for 5 or 40 min at room temperature, respectively. All incubations were performed before the addition of the FRET peptide.
Confocal imaging of FRET peptide
Isolated P. berghei parasites were resuspended in MOPS buffer and plated onto poly-lysine coated plates (200 μL). After 1 min incubation with the FRET peptide (10 μM), plated cells were taken to a confocal microscope (LSM 510, Zeiss) and observed under a 63× objective (water immersion). Cells were excited with UV laser at 351 nm and 364 nm. To select fluorescence, a 375 nm main dichroic mirror was used. Fluorescence was collected with a 385 long-pass dichroic mirror. Images were taken at 3 seconds intervals and ATP (200 μM) was added to the cells after baseline acquisition. Fluorescence was analysed using LSM 510 Image Examiner (Zeiss).
Flow cytometry analysis
Using Flow cytometry analysis viability was assessed in P. yoelii and P. berghei by dihydroethidine (1:200) staining for 20 min at 37°C and analysed by dot plots (side scatter versus fluorescence) of 105 cells. Dihydroethidine (DHT) was excited with a 488 nm Argon laser and fluorescence emission was collected at 518-605 nm. Parameters subject to adjustment of the FACSCalibur flow cytometer were forward scatter (FSC) (log scale, E-1), SSC (log scale, 269), FL-2 (log scale, 505). For all flow cytometry experiments initial gating was carried out with unstained erythrocytes to account for erythrocyte autofluorescence.
Plasmodium berghei infected erythrocytes were incubated in MOPS buffer with Ca2+ (1 mM) and KN-62 (200 μM) or DMSO (0.05%) for 2 h at 37°C. After incubation cells were kept for 2 h at -80°C and subsequently disrupted with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) NP-40 and 0.5% (w/v) sodium deoxycholate) including the phosphatase and protease inhibitors (10 μM NaF, 100 μM orthovanadate, 1 μg ml-1 leupeptin, 1 μg ml-1 pepstatin A, 1 μg ml-1 quimostatin, 100 μg ml-1 benzamidine and 1 mM PMSF) for 30 min at 4°C. Samples were quantified by spectrometry and 10 μg of protein was electrophoresed on an 8% polyacrylamide gel and transferred to nitrocellulose. Mouse monoclonal antibody MAb25.1 [42, 43] (1:1,000) that specifically binds MSP1 was added and incubated overnight at 4°C  After washing, blots were incubated for 2 h with secondary HRP-conjugated anti-mouse IgG antibody (1:10,000, GE Healthcare) and binding was detected using enhanced chemiluminescence.
Results are expressed as mean ± SEM of at least three individual experiments. Student's t-test was used for comparisons between two groups, whereas for repeated measures ANOVA was used for comparisons among larger groups. A P value less than 0.05 was considered indicative of a statistically significant difference. GraphPad Prism software (San Diego, CA, USA) was used for all statistical tests.
Previous studies had shown that extracellular ATP induced a rise of intracellular Ca2+ concentration in P. falciparum parasites. Differences in the modulation of proteolysis by Ca2+ among Plasmodium species indicated the necessity for new comparative studies to clarify the situation[15, 31].
These results indicate that activation of proteolysis by ATP could be triggered by an increase in Ca2+ through purinergic signalling in the rodent malarial parasites. This is better demonstrated by the data in Figures 4C and 4D, where isolated P. berghei and P. yoelii parasites that have been loaded with Fluo4/AM were submitted to different pharmacological treatments; the rise in cytosolic calcium was impaired in the presence of P2X blockers TNP-ATP (50 μM), KN-62 (10 μM), Ip5I (10 μM) and PPADS (50 μM).
ATP is a ubiquitous signalling molecule that recognizes purinergic membrane receptors and modulates several processes studied in a myriad of organisms including slime mould, yeast lizards and mammals (for reviews see [28, 46–51]). Purinergic receptors are divided into two classes namely P1 and P2 (adenosine and ATP/ADP, respectively). The P2 receptor includes two types: P2X (family of ligand-gated ion channel receptors) and P2Y (a family of GPCR) . Despite the presence of purinergic signalling mechanisms in invertebrates and lower eukaryotes the evolutionary distance from parasites to human prevents as molecular identification of the receptor from the genome [53–55]. It is noteworthy that in the parasitic helminth Schistosoma mansoni, a P2X receptor has been identified in the genome database, and using bioinformatic tools Madeira and colleagues identified genes for GPCR-like candidates in the genomes of P. falciparum, P. berghei, P. yoelii and P. chabaudi. As RBCs do not synthesize their own purines de novo, Plasmodium must obtain purine compounds from the extracellular milieu. Endocytosis from the host cell cytosol is thought to be involved in the uptake of nutrients, such as nucleosides, nucleobases and amino acids.
The P. falciparum plasma membrane nucleoside transporter, PfENT1 (P. falciparum equilibrative nucleoside transporter), has been the subject of extensive study [59, 60]. Localized in the plasma membrane  its kinetic parameters and substrate specificity have been determined [59, 60, 62]. The knockout of the PfENT1 gene leads to a reduction in hypoxanthine uptake, and in addition adenosine and inosine transport is affected . The molecular-cellular mechanism by which the parasite obtains extracellular compounds might be related to lipid traffic in the infected RBC  and it is well known that the parasite has the ability to create membranous structures in the infected-RBC and new functions such as the anion channel (new permeation pathway)[65–67].
In the present contribution, it was shown that addition of ATP in a dose dependent manner (Figure 1) to P. berghei and P. yoelii activates intracellular proteolysis. Interestingly, extracellular and intracellular Ca2+ is needed to activate the proteolysis triggered by ATP (Figure 2). It was also found that the purinergic receptor is involved in the ATP signalling pathway to activate the proteases of these rodent malarial parasites since the presence of purinergic antagonists (suramin, PPADS or KN-62) blocked proteolytic activation triggered by ATP in P. berghei and P. yoelii (Figure 3). Differences in the PPADS concentration needed to block protease activity in P. berghei (50 μM) and P. yoelii (100 μM) may be due to differences in the total intracellular Ca2+ concentration mobilized by ATP in both species (Figure 4). This result is in agreement with the presence of a purinergic antagonist (PPADS, TNP-ATP, Ip5I or KN-62) able to inhibit the ability of ATP to induce a rise in calcium in these rodent malarial species (Figure 4). Of interest, the differences in calcium activation of proteolysis between P. berghei and P. yoelii were reported previously.
For the human malaria parasite P. falciparum, merozoite invasion and secondary processing of MSP1 is inhibited by suramin. Here it was shown that MSP1 protein expression and processing in P. berghei parasites is increased by KN-62 (200 μM) treatment (Figure 7) indicating that MSP1 processing may be a downstream effect of the purinergic signalling pathway of P. berghei.
Proteolysis is central to several steps of the Plasmodium life cycle including merozoite invasion and egress from RBC, and haemoglobin digestion. In P. falciparum, blocking purinergic receptors with either KN-62 or Ip51 prevents parasite invasion of RBCs; interestingly the present work showed that KN-62 modulates the P. berghei intracellular cell cycle (Figure 8).
The data presented here support the concept that Plasmodium subverts the host-endocrine system by using extracellular ATP to activate proteolysis to invade or escape from RBCs. The molecular identification of the purinergic receptor in Plasmodium as well as the protease (s) involved in these processes represent the basis of new strategies for development of anti-malarial drugs.
This work was partially supported by grants from Brazilian Agencies FAPESP and CNPq - InBqmed (to C.R.S.G), the UK Medical Research Council (File references: U117532067 and U117532063) and the EU through the Network of Excellence EviMalaR (Health-2009-2.3.2-1-242095). LNC and AB received a FAPESP Fellowship.
- WHO Global Malaria Programme: World Malaria Report 2011. 2011, World Health Organization
- Garcia CR, Takeuschi M, Yoshioka K, Miyamoto H: Imaging Plasmodium falciparum-infected ghost and parasite by atomic force microscopy. J Struct Biol. 1997, 119: 92-98. 10.1006/jsbi.1997.3886.View ArticlePubMed
- Garcia CR, de Azevedo MF, Wunderlich G, Budu A, Young JA, Bannister L: Plasmodium in the postgenomic era: new insights into the molecular cell biology of malaria parasites. Int Rev Cell Mol Biol. 2008, 266: 85-156.View ArticlePubMed
- Goldberg DE, Cowman AF: Moving in and renovating: exporting proteins from Plasmodium into host erythrocytes. Nat Rev Microbiol. 2010, 8: 617-621. 10.1038/nrmicro2420.View ArticlePubMed
- Banerjee R, Liu J, Beatty W, Pelosof L, Klemba M, Goldberg DE: Four plasmepsins are active in the Plasmodium falciparum food vacuole, including a protease with an active-site histidine. Proc Natl Acad Sci USA. 2002, 99: 990-995. 10.1073/pnas.022630099.PubMed CentralView ArticlePubMed
- Coppi A, Tewari R, Bishop JR, Bennett BL, Lawrence R, Esko JD, Billker O, Sinnis P: Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells. Cell Host Microbe. 2007, 2: 316-327. 10.1016/j.chom.2007.10.002.PubMed CentralView ArticlePubMed
- Hall N, Karras M, Raine JD, Carlton JM, Kooij TW, Berriman M, Florens L, Janssen CS, Pain A, Christophides GK: A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses. Science. 2005, 307: 82-86. 10.1126/science.1103717.View ArticlePubMed
- Harris PK, Yeoh S, Dluzewski AR, O'Donnell RA, Withers-Martinez C, Hackett F, Bannister LH, Mitchell GH, Blackman MJ: Molecular identification of a malaria merozoite surface sheddase. PLoS Pathog. 2005, 1: 241-251.View ArticlePubMed
- Rosenthal PJ: Cysteine proteases of malaria parasites. Int J Parasitol. 2004, 34: 1489-1499. 10.1016/j.ijpara.2004.10.003.View ArticlePubMed
- Doerig C, Abdi A, Bland N, Eschenlauer S, Dorin-Semblat D, Fennell C, Halbert J, Holland Z, Nivez MP, Semblat JP: Malaria: targeting parasite and host cell kinomes. Biochim Biophys Acta. 2010, 1804: 604-612.View ArticlePubMed
- Koyama FC, Chakrabarti D, Garcia CR: Molecular machinery of signal transduction and cell cycle regulation in Plasmodium. Mol Biochem Parasitol. 2009, 165: 1-7. 10.1016/j.molbiopara.2009.01.003.PubMed CentralView ArticlePubMed
- Kumar KA, Garcia CR, Chandran VR, Van Rooijen N, Zhou Y, Winzeler E, Nussenzweig V: Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity. Mol Biochem Parasitol. 2007, 156: 32-40. 10.1016/j.molbiopara.2007.07.004.View ArticlePubMed
- Barale JC, Blisnick T, Fujioka H, Alzari PM, Aikawa M, Braun-Breton C, Langsley G: Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase. Proc Natl Acad Sci USA. 1999, 96: 6445-6450. 10.1073/pnas.96.11.6445.PubMed CentralView ArticlePubMed
- Perrin BJ, Huttenlocher A: Calpain. Int J Biochem Cell Biol. 2002, 34: 722-725. 10.1016/S1357-2725(02)00009-2.View ArticlePubMed
- Cruz LN, Alves E, Leal MT, Juliano MA, Rosenthal PJ, Juliano L, Garcia CR: FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii. Int J Parasitol. 2011, 41: 363-372. 10.1016/j.ijpara.2010.10.009.View ArticlePubMed
- Alves E, Bartlett PJ, Garcia CR, Thomas AP: Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells. J Biol Chem. 2011, 286: 5905-5912. 10.1074/jbc.M110.188474.PubMed CentralView ArticlePubMed
- Beraldo FH, Almeida FM, da Silva AM, Garcia CR: Cyclic AMP and calcium interplay as second messengers in melatonin-dependent regulation of Plasmodium falciparum cell cycle. J Cell Biol. 2005, 170: 551-557. 10.1083/jcb.200505117.PubMed CentralView ArticlePubMed
- Gamo FJ, Sanz LM, Vidal J, de Cozar C, Alvarez E, Lavandera JL, Vanderwall DE, Green DV, Kumar V, Hasan S, Brown JR, Peishoff CE, Cardon LR, Garcia-Bustos JF: Thousands of chemical starting points for antimalarial lead identification. Nature. 2010, 465: 305-310. 10.1038/nature09107.View ArticlePubMed
- Krishna S, Pulcini S, Fatih F, Staines H: Artemisinins and the biological basis for the PfATP6/SERCA hypothesis. Trends Parasitol. 2010, 26: 517-523. 10.1016/j.pt.2010.06.014.View ArticlePubMed
- Lew VL, Tiffert T: Is invasion efficiency in malaria controlled by pre-invasion events?. Trends Parasitol. 2007, 23: 481-484. 10.1016/j.pt.2007.08.001.View ArticlePubMed
- Marchesini N, Luo S, Rodrigues CO, Moreno SN, Docampo R: Acidocalcisomes and a vacuolar H + -pyrophosphatase in malaria parasites. Biochem J. 2000, 347: 243-253. 10.1042/0264-6021:3470243.PubMed CentralView ArticlePubMed
- Passos AP, Garcia CR: Characterization of Ca2+ transport activity associated with a non-mitochondrial calcium pool in the rodent malaria parasite. Chabaudi Biochem Mol Biol Int. 1997, 42: 919-925.PubMed
- Sibley LD: Intracellular parasite invasion strategies. Science. 2004, 304: 248-253. 10.1126/science.1094717.View ArticlePubMed
- Vaid A, Sharma P: PfPKB, a protein kinase B-like enzyme from Plasmodium falciparum: II. Identification of calcium/calmodulin as its upstream activator and dissection of a novel signaling pathway. J Biol Chem. 2006, 281: 27126-27133. 10.1074/jbc.M601914200.View ArticlePubMed
- Valderramos SG, Scanfeld D, Uhlemann AC, Fidock DA, Krishna S: Investigations into the role of the Plasmodium falciparum SERCA (PfATP6) L263E mutation in artemisinin action and resistance. Antimicrob Agents Chemother. 2010, 54: 3842-3852. 10.1128/AAC.00121-10.PubMed CentralView ArticlePubMed
- Varotti FP, Beraldo FH, Gazarini ML, Garcia CR: Plasmodium falciparum malaria parasites display a THG-sensitive Ca2+ pool. Cell Calcium. 2003, 33: 137-144. 10.1016/S0143-4160(02)00224-5.View ArticlePubMed
- Srinivasan V, Spence DW, Moscovitch A, Pandi-Perumal SR, Trakht I, Brown GM, Cardinali DP: Malaria: therapeutic implications of melatonin. J Pineal Res. 2010, 48: 1-8. 10.1111/j.1600-079X.2009.00728.x.View ArticlePubMed
- Beraldo FH, Sartorello R, Lanari RD, Garcia CR: Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae). Cell Calcium. 2001, 29: 439-445. 10.1054/ceca.2001.0207.View ArticlePubMed
- Budu A, Peres R, Bueno VB, Catalani LH, Garcia CR: N1-acetyl-N2-formyl-5-methoxykynuramine modulates the cell cycle of malaria parasites. J Pineal Res. 2007, 42: 261-266. 10.1111/j.1600-079X.2006.00414.x.View ArticlePubMed
- Gazarini ML, Thomas AP, Pozzan T, Garcia CR: Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. J Cell Biol. 2003, 161: 103-110. 10.1083/jcb.200212130.PubMed CentralView ArticlePubMed
- Farias SL, Gazarini ML, Melo RL, Hirata IY, Juliano MA, Juliano L, Garcia CR: Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium. Mol Biochem Parasitol. 2005, 141: 71-79. 10.1016/j.molbiopara.2005.01.015.View ArticlePubMed
- Ralevic V, Burnstock G: Postjunctional synergism of noradrenaline and adenosine 5'-triphosphate in the mesenteric arterial bed of the rat. Eur J Pharmacol. 1990, 175: 291-299. 10.1016/0014-2999(90)90567-P.View ArticlePubMed
- Burnstock G, Wood JN: Purinergic receptors: their role in nociception and primary afferent neurotransmission. Curr Opin Neurobiol. 1996, 6: 526-532. 10.1016/S0959-4388(96)80060-2.View ArticlePubMed
- Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA: International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev. 2006, 58: 281-341. 10.1124/pr.58.3.3.PubMed CentralView ArticlePubMed
- Akkaya C, Shumilina E, Bobballa D, Brand VB, Mahmud H, Lang F, Huber SM: The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway. Pflugers Arch. 2009, 457: 1035-1047. 10.1007/s00424-008-0572-8.View ArticlePubMed
- Levano-Garcia J, Dluzewski AR, Markus RP, Garcia CR: Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells. Purinergic Signal. 2010, 6: 365-372. 10.1007/s11302-010-9202-y.PubMed CentralView ArticlePubMed
- Fleck SL, Birdsall B, Babon J, Dluzewski AR, Martin SR, Morgan WD, Angov E, Kettleborough CA, Feeney J, Blackman MJ, Holder AA: Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum. J Biol Chem. 2003, 278: 47670-47677. 10.1074/jbc.M306603200.View ArticlePubMed
- Child MA, Epp C, Bujard H, Blackman MJ: Regulated maturation of malaria merozoite surface protein-1 is essential for parasite growth. Mol Microbiol. 2010, 78: 187-202.PubMed CentralPubMed
- Hirata IY: CMCaCN: Internally quenched fluorogenic protease substrates: solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs. Lett Pept Sci. 1994, 1: 299-308.View Article
- Carmona AK, Juliano MA, Juliano L: The use of Fluorescence Resonance Energy Transfer (FRET) peptides for measurement of clinically important proteolytic enzymes. An Acad Bras Cienc. 2009, 81: 381-392. 10.1590/S0001-37652009000300005.View ArticlePubMed
- Hirata IY, Cezari MH, Nakaie CR, Boschcov P, Ito AS, Juliano MA, Juliano L: Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs. Letters in Peptide Science. 1994, 1: 299-308.View Article
- Freeman RR, Trejdosiewicz AJ, Cross GA: Protective monoclonal antibodies recognising stage-specific merozoite antigens of a rodent malaria parasite. Nature. 1980, 284: 366-368. 10.1038/284366a0.View ArticlePubMed
- Holder AA, Freeman RR: Characterization of a high molecular weight protective antigen of Plasmodium yoelii. Parasitology. 1984, 88 (Pt 2): 211-219.PubMed
- Holder AA, Freeman RR: Immunization against blood-stage rodent malaria using purified parasite antigens. Nature. 1981, 294: 361-364. 10.1038/294361a0.View ArticlePubMed
- Holder AA, Freeman RR: The three major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight precursor. J Exp Med. 1984, 160: 624-629. 10.1084/jem.160.2.624.View ArticlePubMed
- Bagnaresi P, Rodrigues MT, Garcia CR: Calcium signaling in lizard red blood cells. Comp Biochem Physiol A Mol Integr Physiol. 2007, 147: 779-787. 10.1016/j.cbpa.2006.09.015.View ArticlePubMed
- Beraldo FH, Garcia CR: Divergent calcium signaling in RBCs from Tropidurus torquatus (Squamata-Tropiduridae) strengthen classification in lizard evolution. BMC Physiol. 2007, 7: 7-10.1186/1472-6793-7-7.PubMed CentralView ArticlePubMed
- Beraldo FH, Sartorello R, Gazarini ML, Caldeira W, Garcia CR: Red blood cells of the lizards Ameiva ameiva (Squamata, Teiidae) display multiple mechanisms to control cytosolic calcium. Cell Calcium. 2002, 31: 79-87. 10.1054/ceca.2002.0258.View ArticlePubMed
- Burnstock G, Verkhratsky A: Evolutionary origins of the purinergic signalling system. Acta Physiol (Oxf). 2009, 195: 415-447. 10.1111/j.1748-1716.2009.01957.x.View Article
- Allan CY, Fisher PR: In vivo measurements of cytosolic calcium in Dictyostelium discoideum. Methods Mol Biol. 2009, 571: 291-308. 10.1007/978-1-60761-198-1_20.View ArticlePubMed
- Cunningham KW: Acidic calcium stores of Saccharomyces cerevisiae. Cell Calcium. 2011, 50: 129-138. 10.1016/j.ceca.2011.01.010.PubMed CentralView ArticlePubMed
- Burnstock G, Kennedy C: Is there a basis for distinguishing two types of P2-purinoceptor?. Gen Pharmacol. 1985, 16: 433-440. 10.1016/0306-3623(85)90001-1.View ArticlePubMed
- Carlton JM, Angiuoli SV, Suh BB, Kooij TW, Pertea M, Silva JC, Ermolaeva MD, Allen JE, Selengut JD, Koo HL: Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii. Nature. 2002, 419: 512-519. 10.1038/nature01099.View ArticlePubMed
- Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S: Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002, 419: 498-511. 10.1038/nature01097.View ArticlePubMed
- Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD, De La Vega P, Holder AA, Batalov S, Carucci DJ, Winzeler EA: Discovery of gene function by expression profiling of the malaria parasite life cycle. Science. 2003, 301: 1503-1508. 10.1126/science.1087025.View ArticlePubMed
- Raouf R, Blais D, Seguela P: High zinc sensitivity and pore formation in an invertebrate P2X receptor. Biochim Biophys Acta. 2005, 1669: 135-141. 10.1016/j.bbamem.2005.01.009.View ArticlePubMed
- Madeira L, Galante PA, Budu A, Azevedo MF, Malnic B, Garcia CR: Genome-wide detection of serpentine receptor-like proteins in malaria parasites. PLoS One. 2008, 3: e1889-10.1371/journal.pone.0001889.PubMed CentralView ArticlePubMed
- Gherardi A, Sarciron ME: Molecules targeting the purine salvage pathway in Apicomplexan parasites. Trends Parasitol. 2007, 23: 384-389. 10.1016/j.pt.2007.06.003.View ArticlePubMed
- Carter NS, Ben Mamoun C, Liu W, Silva EO, Landfear SM, Goldberg DE, Ullman B: Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparu. J Biol Chem. 2000, 275: 10683-10691. 10.1074/jbc.275.14.10683.View ArticlePubMed
- Parker MD, Hyde RJ, Yao SY, McRobert L, Cass CE, Young JD, McConkey GA, Baldwin SA: Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy. Biochem J. 2000, 349: 67-75. 10.1042/0264-6021:3490067.PubMed CentralView ArticlePubMed
- Rager N, Mamoun CB, Carter NS, Goldberg DE, Ullman B: Localization of the Plasmodium falciparum PfNT1 nucleoside transporter to the parasite plasma membrane. J Biol Chem. 2001, 276: 41095-41099. 10.1074/jbc.M107037200.View ArticlePubMed
- Downie MJ, Saliba KJ, Howitt SM, Broer S, Kirk K: Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1. Mol Microbiol. 2006, 60: 738-748. 10.1111/j.1365-2958.2006.05125.x.View ArticlePubMed
- El Bissati K, Zufferey R, Witola WH, Carter NS, Ullman B, Ben Mamoun C: The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum. Proc Natl Acad Sci USA. 2006, 103: 9286-9291. 10.1073/pnas.0602590103.PubMed CentralView ArticlePubMed
- Murphy SC, Fernandez-Pol S, Chung PH, Prasanna Murthy SN, Milne SB, Salomao M, Brown HA, Lomasney JW, Mohandas N, Haldar K: Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum. Blood. 2007, 110: 2132-2139. 10.1182/blood-2007-04-083873.PubMed CentralView ArticlePubMed
- Ginsburg H, Stein WD: How many functional transport pathways does Plasmodium falciparum induce in the membrane of its host erythrocyte?. Trends Parasitol. 2005, 21: 118-121. 10.1016/j.pt.2005.01.004.View ArticlePubMed
- Kelly JX, Winter RW, Braun TP, Osei-Agyemang M, Hinrichs DJ, Riscoe MK: Selective killing of the human malaria parasite Plasmodium falciparum by a benzylthiazolium dye. Exp Parasitol. 2007, 116: 103-110. 10.1016/j.exppara.2006.12.001.PubMed CentralView ArticlePubMed
- Kirk K, Saliba KJ: Targeting nutrient uptake mechanisms in Plasmodium. Curr Drug Targets. 2007, 8: 75-88. 10.2174/138945007779315560.View ArticlePubMed
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