Patricia Nunes | UFPB - Universidade Federal da Paraíba (original) (raw)
Papers by Patricia Nunes
Magnetic Resonance in Medicine, 2009
Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a sin... more Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single 2H-NMR spectrum is demonstrated with 6-hr and 24-hr fasted rats. Animals were administered [1-2H, 1-13C]glucose, a novel tracer of glucose turnover, and 2H2O. Plasma glucose enrichment from both tracers was quantified by 2H-NMR analysis of monoacetone glucose. The 6-hr fasted group (n = 7) had EGP rates of 95.6 ± 13.3 μmol/kg/min, where 56.2 ± 7.9 μmol/kg/min were derived from PEP; 12.1 ± 2.1 μmol/kg/min from glycerol, and 32.1 ± 4.9 μmol/kg/min from glycogen. The 24-hr fasted group (n = 7) had significantly lower EGP rates (52.8 ± 7.2 μmol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 ± 5.9 μmol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 ± 3.9 and 8.5 ± 1.2 μmol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from 2H2O and 13C-glucose tracers. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc.
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](Nmr in Biomedicine, 2008
The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h... more The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 ± 3% for [13C]bicarbonate perfusion and 91 ± 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 ± 1% and 99 ± 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output. Copyright © 2007 John Wiley & Sons, Ltd.
Nanotoxicology, 2008
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Antimicrobial Agents and Chemotherapy, 2001
As predicted based on structural considerations, we show results indicating that the member of th... more As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1. on March 26, 2014 by guest http://aac.asm.org/ Downloaded from RESULTS ORF YIL120w is an MDR determinant. S. cerevisiae W303.1b deleted for ORF YIL120w did not display any evident VOL. 45, 2001 ORF YIL120w IN YEAST RESISTANCE TO QUINIDINE 1529 on March 26, 2014 by guest http://aac.asm.org/ Downloaded from
Journal of Eukaryotic Microbiology, 2002
ABSTRACT Perkinsus atlanticus cultures were established either with trophozoites isolated from fr... more ABSTRACT 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, IS2, 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.
Biomolecular Engineering, 2003
Two types of iron chelators, desferrioxamine (DFO) and 2,2?-bipyridyl (BIP), selected for their d... more Two 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 mM for DFO and BIP, 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. #
Molecular and Biochemical Parasitology, 2005
We have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and i... more We 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 effect was 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.
Aquaculture, 2005
The protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. Th... more The 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. D
Magnetic Resonance in Medicine, 2009
Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a sin... more Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single 2H-NMR spectrum is demonstrated with 6-hr and 24-hr fasted rats. Animals were administered [1-2H, 1-13C]glucose, a novel tracer of glucose turnover, and 2H2O. Plasma glucose enrichment from both tracers was quantified by 2H-NMR analysis of monoacetone glucose. The 6-hr fasted group (n = 7) had EGP rates of 95.6 ± 13.3 μmol/kg/min, where 56.2 ± 7.9 μmol/kg/min were derived from PEP; 12.1 ± 2.1 μmol/kg/min from glycerol, and 32.1 ± 4.9 μmol/kg/min from glycogen. The 24-hr fasted group (n = 7) had significantly lower EGP rates (52.8 ± 7.2 μmol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 ± 5.9 μmol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 ± 3.9 and 8.5 ± 1.2 μmol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from 2H2O and 13C-glucose tracers. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc.
Nmr in Biomedicine, 2008
The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h... more The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 ± 3% for [13C]bicarbonate perfusion and 91 ± 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 ± 1% and 99 ± 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output. Copyright © 2007 John Wiley & Sons, Ltd.
Nanotoxicology, 2008
Publication details, including instructions for authors and subscription information:
Antimicrobial Agents and Chemotherapy, 2001
As predicted based on structural considerations, we show results indicating that the member of th... more As predicted based on structural considerations, we show results indicating that the member of the major facilitator superfamily encoded by Saccharomyces cerevisiae open reading frame YIL120w is a multidrug resistance determinant. Yil120wp was implicated in yeast resistance to ketoconazole and quinidine, but not to the stereoisomer quinine; the gene was thus named QDR1. Qdr1p was proved to alleviate the deleterious effects of quinidine, revealed by the loss of cell viability following sudden exposure of the unadapted yeast population to the drug, and to allow the earlier eventual resumption of exponential growth under quinidine stress. However, QDR1 gene expression had no detectable effect on the susceptibility of yeast cells previously adapted to quinidine. Fluorescence microscopy observation of the distribution of the Qdr1-green fluorescent protein fusion protein in living yeast cells indicated that Qdr1p is a plasma membrane protein. We also show experimental evidence indicating that yeast adaptation to growth with quinidine involves the induction of active expulsion of the drug from preloaded cells, despite the fact that this antiarrhythmic and antimalarial quinoline ring-containing drug is not present in the yeast natural environment. However, we were not able to prove that Qdr1p is directly implicated in this export. Results clearly suggest that there are other unidentified quinidine resistance mechanisms that can be used in the absence of QDR1. on March 26, 2014 by guest http://aac.asm.org/ Downloaded from RESULTS ORF YIL120w is an MDR determinant. S. cerevisiae W303.1b deleted for ORF YIL120w did not display any evident VOL. 45, 2001 ORF YIL120w IN YEAST RESISTANCE TO QUINIDINE 1529 on March 26, 2014 by guest http://aac.asm.org/ Downloaded from
Journal of Eukaryotic Microbiology, 2002
ABSTRACT Perkinsus atlanticus cultures were established either with trophozoites isolated from fr... more ABSTRACT 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, IS2, 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.
Biomolecular Engineering, 2003
Two types of iron chelators, desferrioxamine (DFO) and 2,2?-bipyridyl (BIP), selected for their d... more Two 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 mM for DFO and BIP, 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. #
Molecular and Biochemical Parasitology, 2005
We have exploited the experimental accessibility of the protozoan parasite Perkinsus olseni and i... more We 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 effect was 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.
Aquaculture, 2005
The protozoan parasite Perkinsus olseni causes severe losses among Ruditapes decussatus clams. Th... more The 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. D