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- Estudos sobre aproveitamento biotecnológico da quitina produzida por organismos marinhosPublication . Afonso, R.; Fonseca, Maria Leonor Quintais Cancela da; Gama, Francisco Miguel Portela daO oceano é um dos mais ricos habitats do planeta, mas também um dos menos explorados, embora a sua grande diversidade ofereça enormes potencialidades na área da biotecnologia. A quitina, o segundo polissaccarídeo mais abundante a seguir à celulose, é encontrada principalmente no exosqueleto de crustáceos e parede celular de fungos. As suas aplicações biotecnológicas são vastas, mas devido à dificuldade da sua extracção de maneira reprodutível e sob condições económicas viáveis, o seu uso é limitado. Com o objectivo de verificar a potencialidade do parasita marinho Perkinsus olseni como fonte de quitina, a cultura in vitro deste parasita, préviamente estabelecida a partir de amêijoas (Ruditapes decussatus) infectadas provenientes da Ria Formosa, foi optimizada com sucesso em meio DMEM:Ham’s F-12 (1:2) e a sua propagação em maior escala foi conseguida com uma produção de biomassa de 2,1 mg/L/h. A quitina, um dos componentes da parede celular de P. olseni, foi isolada através de dois métodos distintos: (i) por extracção alcalina em diferentes condições e (ii) por extracção com o solvente DMAC/LiCl 5%. O material extraído foi inicialmente caracterizado por FTIR, após o qual foram escolhidas duas amostras, uma extraída pelo método alcalino (qAlc) e outra extraída com solvente (qDMAC). Prosseguiu-se então com a caracterização das duas amostras através de análise elementar, quantificação de proteína residual e acetilglucosamina, difracção de raios-x e RMN de 13C do estado sólido. A caracterização foi algo inconclusiva, mas pareceu indicar a presença de uma β-quitina amorfa.
- Shikimate and folate pathways in the protozoan parasite, Perkinsus olseniPublication . Elandalloussi, Laurence M.; Rodrigues, Pedro; Afonso, Ricardo; Leite, Ricardo; Nunes, Patrícia A.; Cancela, LeonorWe have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and its similarities to apicomplexan parasites to investigate the influence of specific drugs on its proliferation. For this purpose, shikimate and folate pathways present an attractive target for parasitic therapy given their major differences with mammalian pathways. Glyphosate, a potent inhibitor of the shikimate pathway enzyme EPSP synthase inhibited the in vitro proliferation of P. olseni in a dose-dependent manner and this effectwas reversed by addition of chorismate, indicating the presence of a shikimate pathway. However, this effect was not antagonised by p-aminobenzoate or folic acid. Furthermore, antagonism was observed, via pyrimethamine to glyphosate inhibitory effect, suggesting that the shikimate pathway is not essential for the biosynthesis of folate precursors and is therefore crucial for another pathway downstream from chorismate. In addition, sulfadiazine, a well known inhibitor of dihydropteorate synthase, an enzyme of the folate biosynthetic pathway,had no inhibitory effect on P. olseni proliferation. In view of these results, the parasite does not appear to require the folate biosynthesis pathway for its survival and is most likely able to use exogenous folate. Even though pyrimethamine was found to inhibit P. atlanticus growth, this inhibitory effect could not be reversed by co-addition of folic acid. Therefore, we propose that the effect of pyrimethamine observed in this study results from the inhibition of a target other than dihydrofolate reductase. Similarly, proguanil target is likely to be separate from DHFR since only its metabolite cycloguanil has been shown to have inhibitory properties on DHFR.
- A microelectrode impedance method to measure interaction of cellsPublication . Gomes, Henrique L.; Leite, Ricardo; Afonso, R.; Stallinga, Peter; Cancela, M. LeonorAn impedance method was developed to determine how immune system cells (hemocyte) interact with intruder cells (parasites). When the hemocyte cells interact with the parasites, they cause a defensive reaction and the parasites start to aggregate in clusters. The level of aggregation is a measure of the host-parasite interaction, and provides information about the efficiency of the immune system response. The cell aggregation is monitored using a set of microelectrodes. The impedance spectrum is measured between each individual microelectrode and a large reference electrode. As the cells starts to aggregate and settle down towards the microelectrode array the impedance of the system is changed. It is shown that the system impedance is very sensitive to the level of cell aggregation and can be used to monitor in real time the interaction between hemocyte cells and parasites.
- Peroxides with antiplasmodial activity inhibit proliferation of Perkinsus olseni, the causative agent of Perkinsosis in bivalvesPublication . Araujo, Nuna C. P.; Afonso, Ricardo; Bringela, A.; Cancela, Leonor; Cristiano, Maria Lurdes Santos; Leite, RicardoPerkinsus olseni, the causative agent of Perkinsosis, can drastically affect the survival of target marine mollusks, with dramatic economic consequences for aquaculture. P. olseni is a member of the Alveolata group, which also comprises parasites that are highly relevant for medical and veterinary sciences such as Plasmodium falciparum and Toxoplasma. P. olseni shares several unique metabolic pathways with those pathological parasites but is not toxic to humans. In this work, six antimalarially active peroxides, derived from the natural product artemisinin or synthetic trioxolanes, were synthesized and tested on P. olseni proliferation and survival. All peroxides tested revealed an inhibitory effect on P. olseni proliferation atmicromolar concentrations. The relevance of the peroxide functionality on toxicity and the effect of Fe(II)-intracellular concentration on activity were also evaluated. Results demonstrated that the peroxide functionality is the toxofore and intracellular iron concentration also proved to be a crucial co-factor on the activation of peroxides in P. olseni. These data points to a mechanismof bioactivation in P. olseni sharing similaritieswith the one proposed in P. falciparumparasites. Preliminary studies on bioaccumulation were conducted using fluorescent-labeled peroxides. Results show that synthetic trioxolanes tend to accumulate on a vacuolewhile the labeled artemisinin accumulates in the cytoplasm. Preliminary experiments on differential genes expression associated to Fe(II) transport protein (Nramp) and calcium transport protein (ATP6/SERCA) were also conducted by qPCR. Results point to a fourfold increase in expression of both genes upon exposure to trioxolanes and approximately twofold upon exposure to artemisinin derivatives. Data obtained in this investigation is relevant for better understanding of the biology of Perkinsus andmay also be important in the development of new strategies for Perkinsosis prevention and control.
- Effect of desferrioxamine and 2,2 '-bipyridyl on the proliferation of Perkinsus atlanticusPublication . Elandalloussi, L. M.; Afonso, R.; Nunes, Patrícia A.; Cancela, LeonorTwo types of iron chelators, desferrioxamine (DFO) and 2,2-bipyridyl (BIP), selected for their differential binding properties, permeability and stoechiometry, were tested for their ability to inhibit the in vitro proliferation of the carpet shell clam parasite Perkinsus atlanticus. A tetrazolium-based assay was used to determine the effect of the drugs on cell proliferation. Both chelators were able to inhibit P. atlanticus proliferation in a dose-dependent manner, the 50% inhibitory concentration were 14 and 24 VM for DFO and BIR respectively, in a 72 h test. This effect was reversed by co-addition of iron, confirming that this activity is due to the sequestration of iron. These results indicate a high degree of susceptibility of the protozoan parasite to chelator-induced iron deprivation. However, this effect was reversible upon removal of the drugs, indicating that the action of both chelators was cytostatic. For the range of concentrations tested the combined drug effects was not significantly higher than the additive effect of the individual drugs. (C) 2003 Elsevier B.V. All rights reserved.
- Effect of antiprotozoal drugs on the proliferation of the bivalve parasite Perkinsus olseniPublication . Elandalloussi, L. M.; Leite, Ricardo; Rodrigues, Pedro; Afonso, R.; Nunes, Patrícia A.; Cancela, LeonorThe protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. The development of an in vitro culture of this parasite together with the use of a proliferation assay has provided the opportunity to screen for drug sensitivity of this parasite. Xenobiotics known for their antimalarial and antiprotozoal properties were tested against P. olseni. Only four of these drugs, namely cycloheximide, pyrimethamine, deferoxamine (DFO) and 2,2-bipyridyl (BIP), showed in vitro inhibitory effect on the parasite proliferation. Two in vivo experiments were designed to determine the effect of iron chelators on reducing P. olseni infection in clams. For this purpose, naturally infected clams from the Ria Formosa, Portugal, were exposed to DFO and BIP at various concentrations. In the first experiment, hemolymph samples were taken from each clam before and after treatment to determine the infection intensity and in the second experiment, clams were randomly distributed in groups of five and the parasite burden in treated and untreated groups was determined at the end of the experiment on the whole clam wet tissues. Only DFO was found to be effective in reducing in vivo P. olseni infections. In addition, acute toxicity of DFO and BIP has been determined and no mortality of Perkinsus-free clams was observed. (C) 2004 Elsevier B.V. All rights reserved.
- Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscsPublication . Elandalloussi, L. M.; Leite, Ricardo; Afonso, R.; Nunes, Patrícia A.; Robledo, J. A. F.; Vasta, G. R.; Cancela, LeonorPerkinsus atlanticus and P. marinus have been associated with mass mortality of bivalve molluscs. Perkinsus infections are routinely diagnosed by histology or the fluid thioglycollate medium (FTM) assay. In this study, we describe the development of a PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid detection of Perkinsus species. The PCR reactions were selected to either amplify an IGS sequence region shared by currently accepted Perkinsus species or to simultaneously amplify IGS regions specific to either P. atlanticus or P. marinus. The specific hybridisation of DIG-labelled amplified products to species-specific capture probes was detected colorimetrically. This assay is able to specifically detect P. atlanticus and P. marinus, and the intensity of the colorimetric signal is dependent upon the amount of amplified product. The PCR-ELISA assay format is 100-fold more sensitive than visualisation of PCR products on ethidium bromide (EtdBr)-stained agarose gels, and as sensitive as Southern hybridisation. The sensitivity limit of PCR-ELISA was 1 pg of DNA front P. atlanticus. No cross-reactivity of the assay was observed against the host DNA. When applied to the detection of P. atlanticus in clams, 39 samples out of 45 yielded concordant results for FTM assay and PCR-ELISA detection. (C) 2003 Elsevier Ltd. All rights reserved.
- Effect of the herbicide Roundup® on Perkinsus olseni in vitro proliferation and in vivo survival when infecting a permissive host, the Clam Ruditapes decussatusPublication . Elandalloussi, Laurence M.; Leite, Ricardo; Rodrigues, Pedro; Afonso, Ricardo; Cancela, LeonorCoastal habitats are increasingly being exposed to herbicide contamination from urban and agricultural catchments. Data on its toxicity on aquatic ecosystems, especially those based on sediment, are relatively scarce. This study aimed at investigating whether the susceptibility of an aquatic filter-feeding organism, the carpet-shell clam (Ruditapes decussatus) to the parasite Perkinsus olseni was influenced by the herbicide Roundup and its active ingredient glyphosate. The effect of Roundup and glyphosate on P. olseni in vitro proliferation was also evaluated and appeared to confirm the higher toxicity of Roundup when compared with technical grade glyphosate.
- Effect of antiprotozoal drugs on the proliferation of the bivalve parasite Perkinsus olseniPublication . Elandalloussi, Laurence M.; Leite, Ricardo; Rodrigues, Pedro; Afonso, Ricardo; Nunes, Patrícia A.; Cancela, LeonorThe protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. The development of an in vitro culture of this parasite together with the use of a proliferation assay has provided the opportunity to screen for drug sensitivity of this parasite. Xenobiotics known for their antimalarial and antiprotozoal properties were tested against P. olseni. Only four of these drugs, namely cycloheximide, pyrimethamine, deferoxamine (DFO) and 2,2-bipyridyl (BIP), showed in vitro inhibitory effect on the parasite proliferation. Two in vivo experiments were designed to determine the effect of iron chelators on reducing P. olseni infection in clams. For this purpose, naturally infected clams from the Ria Formosa, Portugal, were exposed to DFO and BIP at various concentrations. In the first experiment, hemolymph samples were taken from each clam before and after treatment to determine the infection intensity and in the second experiment, clams were randomly distributed in groups of five and the parasite burden in treated and untreated groups was determined at the end of the experiment on the whole clam wet tissues. Only DFO was found to be effective in reducing in vivo P. olseni infections. In addition, acute toxicity of DFO and BIP has been determined and no mortality of Perkinsus-free clams was observed.
- Perkinsus Atlanticus – desenvolvimento de um método de diagnóstico para detecção de infecção em moluscos bivalvesPublication . Leite, Ricardo; Rodrigues, Pedro; Elandalloussi, Laurence M.; Afonso, Ricardo; Nunes, Patrícia A.; Cancela, LeonorDiferentes espécies do género Perkinsus, um protozoário parasita pertencente ao novo phylum Perkinsea, podem ser encontradas mundialmente e constituem uma séria ameaça aos moluscos bivalves como as ostras, amêijoas, abalones e vieiras, os quais têm um papel importante não só do ponto de vista comercial, mas também ecológico.