Browsing by Author "Nunes, Patrícia A."
Now showing 1 - 10 of 13
Results Per Page
Sort Options
- AQR1 gene (ORF YNL065w) encodes a plasma membrane transporter of the major facilitator superfamily that confers resistance to short-chain monocarboxylic acids and quinidine in Saccharomyces cerevisiaePublication . Tenreiro, S.; Nunes, Patrícia A.; Viegas, C. A.; Neves, M. S.; Teixeira, M. C.; Cabral, M. G.; Sa Correia, I.We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C-2-C-6), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution. (C) 2002 Elsevier Science (USA).
- Batch operational stability of immobilized heterologous Rhizopus oryzae lipase during acidolysis of virgin olive oil with medium-chain fatty acidsPublication . Nunes, Patrícia A.; Cabral, Paula Pires; Guillen, M.; Valero, F.; Ferreira Dias, S.Structured triacylglycerols containing medium-chain fatty acids (M) at positions sn-1,3 and long-chain fatty acids (L) at the sn-2 position (MLM type), were obtained by acidolysis of virgin olive oil with caprylic or capric acid, in solvent-free media, at 40 degrees C, catalyzed by a heterologous Rhizopus oryzae lipase (r-ROL) immobilized in Eupergir (R) C or in Lewatit VP OC 1600. The biocatalyst immobilized in Eupergit was reused in consecutive 23-h batches with rehydration of the biocatalyst between batches. A first-order deactivation was observed (half-life time, t(1/2) = 39.0 h for caprylic; t(1/2) = 54.3 h for capric acid). During acidolysis with capric acid catalyzed by r-ROL immobilized in Lewatit VP OC 1600, without rehydration, a first-order deactivation was observed (t(1/2) = 49.1 h); with rehydration, a considerable increase in stability was observed (t(1/2) = 234 h; Sadana's series-type inactivation kinetics model). (C) 2012 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.
- Development of an in vitro clonal culture and characterization of the rRNA gene cluster of Perkinsus atlanticus, a protistan parasite of the clam Tapes decussatusPublication . Robledo, J. A. F.; Nunes, Patrícia A.; Cancela, Leonor; Vasta, G. R.Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter-cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3-100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally-cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus "genus-specific" PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.
- 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.
- 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.
- 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.
- Microbial quality of edible seeds commercially available in southern PortugalPublication . Silva, Daniela; Nunes, Patrícia A.; Melo, Jessie; Quintas, CéliaIn the present work, the microbiological quality of sesame, flaxseed, chia, pumpkin sunflower seeds, a mix of seeds, as well as flaxseed flour, marketed in southern Portugal, were studied through the counting of aerobic microorganisms at 30 degrees C (AM), molds and yeast (M&Y), Escherichia coli (beta-glucuronidase positive) (beta-GP E. coli), Staphylococcus coagulase positive, and detection of Salmonella spp. The persistence of AM and M&Y populations were also counted in organic and non-organic flaxseed at 20 degrees C for 11 months. The seeds with the highest average of AM were flaxseed (1,3 x 10(6) CFU/g) followed by flaxseed flour (1,1 x 10(6) CFU/g) while the lowest level was found in chia (2,9 x 10(4) CFU/g). This seed also presented the lowest average values of filamentous fungi (9,8 x 10(2) CFU/g), whereas sunflower seeds had the highest levels (1,7 x 10(5) CFU/g). Flaxseed flour had the highest yeast counts (1,5 x 10(4) CFU/g). Although some samples had high levels of AM and fungi, beta-GP E. coli and Salmonella were not detected, therefore, they complied with the microbiological criteria of the European Union. The organic flaxseed contained higher numbers of AM and M&Y than the non-organic ones (p < 0.05). In addition, the storage of flaxseed at 20 degrees C resulted in changes of AM and M&Y, showing that these populations were able to remain viable after eleven months (AM Log 5.4-Log 5.6; M&Y Log 2.8 - Log 4.1). The results obtained in the present study, namely those high levels of AM and fungi (>10(6) and 10(4) CFU/g respectively), alert to the need of improving processing practices, storage/distribution conditions of edible seeds and derivatives, as well as the requirement of implementing adequate decontamination techniques.
- Optimized production of MLM Triacylglycerols catalyzed by immobilized heterologous rhizopus oryzae lipasePublication . Nunes, Patrícia A.; Cabral, Paula Pires; Guillen, M.; Valero, F.; Dias, S. FerreiraResponse surface methodology was used to model and optimize the acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, aimed at the production of low caloric triacylglycerols (TAG) of MLM type, in solvent free media, catalyzed by the heterologous Rhizopus oryzae lipase (r-ROL) immobilized in Eupergit(A (R)) C. This lipase was produced in the methylotrophic yeast Pichia pastoris Mut(s) phenotype (experiments with C10:0) or a Mut(+) phenotype (experiments with C8:0), under different operational conditions. The r-ROL used in experiments with C10:0 presented a hydrolytic activity about 5 times of that presented by r-ROL used in acidolysis with C8:0. The experiments were carried out following a central composite rotatable design, as a function of the molar ratio (MR) medium chain fatty acid/TAG (1.6-4.4) and temperature (25-55 A degrees C). Convex surfaces described by second order polynomials as a function of MR and temperature were well fitted to fatty acid incorporation values. After 24-h reaction, the predicted maximum incorporation of caprylic (15.5 mol%) or capric (33.3 mol%) acids in olive oil occurs at 37 and 35 A degrees C, respectively, and at C8:0/TAG of 2.8:1 or C10:0/TAG of 3:1. These predicted optima were experimentally validated. Fermentation conditions used in r-ROL production highly affected hydrolytic activity and to a lesser extent interesterification activity.
- 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.