Publications by authors named "Patrícia Duarte"

27 Publications

  • Page 1 of 1

Metabolic Profile Changes in Mangalarga Marchador Horses Subjected to A Hypercaloric Diet Evaluated by Proton NMR Spectroscopy.

J Equine Vet Sci 2021 Aug 11;103:103684. Epub 2021 Jun 11.

Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

The incidence of equine metabolic syndrome, a condition that results in endocrinopathic laminitis, is increasing worldwide. Although it is well known that the development of this syndrome depends on imbalances in energy metabolism and genetic traits, its pathophysiology remains unclear. Hydrogen nuclear magnetic resonance (H NMR) is a powerful tool used to compare metabolic profiles and to discriminate metabolites in living beings. The aim was to apply this technology to detect blood biomarkers candidates in horses that were previously demonstrated to developed metabolic changes when subjected to induced obesity. Nine Mangalarga Marchador horses received a hypercaloric diet for 5 months and serum metabolomic analysis was performed before, during, and after the diet period. The H NMR results were subjected to multivariate analysis and NMR analysis allowed to identify six compounds (alanine, threonine, choline, α-glucose, β-glucose, and creatinine), and observe the increasing choline level over the assessment period in four animals. A hypercaloric diet altered the metabolic profile of horses, with an individual bias in the time at which these changes occurred. This study is the first to describe metabolomic compounds in Mangalarga Marchador horses subjected to a hypercaloric diet rich in non-structural carbohydrates.
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http://dx.doi.org/10.1016/j.jevs.2021.103684DOI Listing
August 2021

Molecular similarity between the mechanisms of epithelial fusion and fetal wound healing during the closure of the caudal neural tube in mouse embryos.

Dev Dyn 2021 Jul 10;250(7):955-973. Epub 2021 Feb 10.

Neurodevelopment Research Group, Institute of Biomedicine of Seville (IBiS)/Hospital Virgen del Rocio/US/CSIC, Sevilla, Spain.

Background: Neural tube (NT) closure is a complex developmental process that takes place in the early stages of embryogenesis and that is a key step in neurulation. In mammals, the process by which the neural plate generates the NT requires organized cell movements and tissue folding, and it terminates with the fusion of the apposed ends of the neural folds.

Results: Here we describe how almost identical cellular and molecular machinery is used to fuse the spinal neural folds as that involved in the repair of epithelial injury in the same area of the embryo. For both natural and wound activated closure of caudal neural tissue, hyaluronic acid and platelet-derived growth factor signaling appear to be crucial for the final fusion step.

Conclusions: There seems to be no general wound healing machinery for all tissues but rather, a tissue-specific epithelial fusion machinery that embryos activate when necessary after abnormal epithelial opening.
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http://dx.doi.org/10.1002/dvdy.306DOI Listing
July 2021

Biological properties of functional flavoring produced by enzymatic esterification of citronellol and geraniol present in essential oil.

Nat Prod Res 2020 Aug 25:1-7. Epub 2020 Aug 25.

Food Engineering Department, Universidade Regional Integrada do Alto Uruguai e das Missões-URI Erechim, Erechim, RS, Brazil.

The chemical composition and biological properties of citronella essential oil were modified by enzymatic esterification reaction of the major monoterpenic alcohols with cinnamic acid. The almost complete conversion of geraniol and citronellol present in the citronella () essential oil, into geranyl (99%) and citronellyl (98%) cinnamates was obtained after 48 hours of reaction using a molar ratio of 3:1 (cinnamic acid/alcohol), lipase concentration (Novozym 435) of 15% (w/w) and 70 °C. The esterified oil showed higher antimicrobial activity against bacteria resistant to oxacillin and penicillin and also greater larvicidal activity against larvae compared to unesterified oil. The results concerning the evaluation of toxicity against and cytotoxicity against monkey kidney epithelial cells also showed the superiority of the esterified oil.
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http://dx.doi.org/10.1080/14786419.2020.1810032DOI Listing
August 2020

Outcomes from an international multicenter registry of patients with acute gastrointestinal bleeding undergoing endoscopic treatment with Hemospray.

Dig Endosc 2020 Jan 30;32(1):96-105. Epub 2019 Aug 30.

Division of Surgery and Interventional Science, University College London (UCL), London, UK.

Background And Aim: Acute gastrointestinal bleeding carries poor outcomes unless prompt endoscopic hemostasis is achieved. Mortality in these patients remains significant. Hemospray is a novel intervention that creates a mechanical barrier over bleeding sites. We report the largest dataset of patient outcomes after treatment with Hemospray from an international multicenter registry.

Patients And Methods: Prospective data (Jan 2016-May 2018) from 12 centers across Europe were collected. Immediate hemostasis was defined as endoscopic cessation of bleeding within 5 min after application of Hemospray. Rebleeding was defined as subsequent drop in hemoglobin, hematemesis, persistent melena with hemodynamic compromise post-therapy.

Results: Three hundred and fourteen cases were recruited worldwide (231 males, 83 females). Median pretreatment Blatchford score was 11 (IQR: 8-14) and median complete Rockall score (RS) was 7 (IQR: 6-8) for all patients. Peptic ulcer disease (PUD) was the most common pathology (167/314 = 53%) and Forrest Ib the most common bleed type in PUD (100/167 = 60%). 281 patients (89.5%) achieved immediate hemostasis after successful endoscopic therapy with Hemospray. Rebleeding occurred in 29 (10.3%) of the 281 patients who achieved immediate hemostasis. Seven-day and 30-day all-cause mortality were 11.5% (36/314) and 20.1% (63/314), respectively (lower than the predicted rates as per the RS). Similar hemostasis rates were noted in the Hemospray monotherapy (92.4%), combination therapy (88.7%) and rescue therapy (85.5%) groups.

Conclusions: These data show high rates of immediate hemostasis overall and in all subgroups. Rebleeding and mortality rates were in keeping/lower than predicted rates.
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http://dx.doi.org/10.1111/den.13502DOI Listing
January 2020

Bioremediation of bezafibrate and paroxetine by microorganisms from estuarine sediment and activated sludge of an associated wastewater treatment plant.

Sci Total Environ 2019 Mar 20;655:796-806. Epub 2018 Nov 20.

CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Porto, Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal.

The present work aimed to explore the potential of autochthonous microorganisms from an urban estuary and from activated sludge of an associated wastewater treatment plant (WWTP), for biodegradation of an antidepressant drug, paroxetine, and on a cholesterol-lowering agent, bezafibrate. These compounds were chosen as representatives of extensively used pharmaceuticals. Autochthonous microorganisms from the indicated sources were exposed to the target pharmaceuticals (1 mg/L) in co-metabolism with sodium acetate (500 mg/L) along a two-weeks period, for a total of 7 two-weeks periods (here referred as cycles). Exposures were carried out in batch mode, under different incubation conditions (agitation vs. static). Removal of pharmaceuticals was monitored at the end of each cycle, by analysing the culture medium. For paroxetine, fluoride ion release was also followed as an indicator of defluorination of the molecule. The structure of the bacterial communities was analysed by ARISA (Automated rRNA Intergenic Spacer Analysis), at the beginning of the experiment and at the end of the first and the last cycles to identify substantial changes associated with the time of exposure, the incubation conditions and the presence and type of pharmaceuticals. Incubation conditions affected not only the bacterial community structure, but also the biodegradation efficiency. At the beginning of the experiment, removal of target pharmaceuticals was found to be lower under agitation than under static conditions, but at the end of the experiment, results showed high removal of the pharmaceuticals from the culture medium (>97%) under both conditions, mainly by microbiological processes. For paroxetine, adsorption and abiotic processes also had an important influence on its removal, but defluorination only occurred in the presence of microorganisms. These results highlight that autochthonous microorganisms from estuarine sediments and WWTP sludge have high ability to remove the selected pharmaceuticals with relevant implications for the development of new bioremediation tools for environmental restoration.
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http://dx.doi.org/10.1016/j.scitotenv.2018.11.285DOI Listing
March 2019

The Three-Factor Eating Questionnaire-R21: a confirmatory factor analysis in a Portuguese sample.

Eat Weight Disord 2020 Feb 28;25(1):247-256. Epub 2018 Aug 28.

CINEICC, Center for Research in Neuropsychology and Cognitive Behavioral Intervention, Faculty of Psychology and Education Sciences, University of Coimbra, Rua do Colégio Novo, Apartado 6153, 3001-802, Coimbra, Portugal.

The Three-Factor Eating Questionnaire (TFEQ) is one of the most widely used instruments to study different eating behaviors. It measures three types of eating behaviors namely: cognitive restraint, uncontrolled eating and emotional eating. The present study aims to evaluate the factor structure and reliability of the Portuguese version of the TFEQ-R21, using a confirmatory factor analysis (CFA). The sample includes 468 participants from the general population, with ages ranging from 18 to 60 years. Results from the CFA confirmed the TFEQ-R21 three-factor structure and the model revealed an acceptable fit to the data (χ(186) = 443.211, p < 0.001; χ/df = 2.329; CFI = 0.933; TLI = 0.925; RMSEA = 0.054; SMRS = 0.053). Multi-group analysis results support strong measurement invariance across genders. Furthermore, all three dimensions presented adequate psychometric properties. Overall, results support that the Portuguese version of the TFEQ-R21 is a useful, reliable and robust instrument to assess relevant eating behaviors.Level of evidence V, descriptive studies.
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http://dx.doi.org/10.1007/s40519-018-0561-7DOI Listing
February 2020

Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

Methods Mol Biol 2018 ;1789:81-99

CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H-ATPase and the V-H-pyrophosphatase, also producing further information on vacuole purity.
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http://dx.doi.org/10.1007/978-1-4939-7856-4_7DOI Listing
February 2019

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

Biotechnol Bioeng 2018 03 11;115(3):630-644. Epub 2017 Dec 11.

iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs.
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http://dx.doi.org/10.1002/bit.26504DOI Listing
March 2018

Caudal epidural anesthesia in mares after bicarbonate addition to a lidocaine-epinephrine combination.

Vet Anaesth Analg 2017 Jul 11;44(4):943-950. Epub 2017 Jan 11.

Department of Veterinary Clinics and Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Electronic address:

Objective: To investigate the nociceptive and clinical effects of buffering a lidocaine-epinephrine solution with sodium bicarbonate in caudal epidural block in mares.

Study Design: Prospective randomized controlled trial.

Animals: Six mixed-breed mares weighing 350-440 kg.

Methods: Each animal was administered two caudal epidural injections, 72 hours apart, using different solutions prepared immediately before injection. The control solution was 7 mL 2% lidocaine hydrochloride with epinephrine hemitartrate (1:200,000) added to 3 mL sterile water for injection (pH 2.9). The alkalinized solution was 7 mL of lidocaine-epinephrine solution added to 2.3 mL sterile water for injection and 0.7 mL 8.4% sodium bicarbonate (pH 7.4). Nociception was evaluated by response to skin pinching at 31 sites in the sacral region and around the perimeter of the anogenital area (distances of 10, 15 and 20 cm) before, and 5, 10 and 15 minutes after epidural injection, then every 15 minutes until the return of nociception in all evaluated sites. The onset and duration times, and intensity of ataxia (grades 0 to 3) were recorded. The paired t test was used to compare the onset and duration of anesthesia and ataxia (p<0.05).

Results: Alkalization of the solution resulted in significant decreases in the average time of onset of loss of nociception in the sacral region (40%) and around the perimeter of the anogenital area extending up to 5 cm (36%) and from 5 to 10 cm (32%) from the anus and vulva. Alkalization also decreased the average duration of ataxia (33%), without affecting the duration and extent of anesthesia or the degree of ataxia.

Conclusions And Clinical Relevance: Alkalization of lidocaine-epinephrine solution is advantageous in shortening the duration of ataxia and hastening the onset of anesthesia in areas adjacent to the anogenital area, without reducing the duration of epidural anesthesia, in mares.
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http://dx.doi.org/10.1016/j.vaa.2016.11.003DOI Listing
July 2017

Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells.

Sci Rep 2017 08 17;7(1):8590. Epub 2017 Aug 17.

iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, Oeiras, 2780-901, Portugal.

The immature phenotype of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) constrains their potential in cell therapy and drug testing. In this study, we report that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. Integrated "-Omics" analyses showed that addition of galactose to culture medium improves total oxidative capacity of the cells and ameliorates fatty acid oxidation avoiding the lipotoxicity that results from cell exposure to high fatty acid levels. This study provides an important link between substrate utilization and functional maturation of hPSC-CMs facilitating the application of this promising cell type in clinical and preclinical applications.
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http://dx.doi.org/10.1038/s41598-017-08713-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561128PMC
August 2017

Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

Plant Physiol 2016 08 29;171(4):2371-8. Epub 2016 Jun 29.

CIBIO/InBIO-Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, 4485-661 Vairão, Portugal (S.B., T.M.-C., J.G.G., M.S.); Instituto de Investigação e Inovação em Saúde, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal (I.C., A.L.G., P.D.);Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal (I.C., M.S.);Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal (R.G., T.L., C.A., C.B., N.P.M.);REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal (P.A., P.V.); andREQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal (I.M.V., J.A.R.)

Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus.
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http://dx.doi.org/10.1104/pp.16.01028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972299PMC
August 2016

Protoplast Transformation as a Plant-Transferable Transient Expression System.

Methods Mol Biol 2016 ;1405:137-48

Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

The direct uptake of DNA by naked plant cells (protoplasts) provides an expression system of exception for the quickly growing research in non-model plants, fuelled by the power of next-generation sequencing to identify novel candidate genes. Here, we describe a simple and effective method for isolation and transformation of protoplasts, and illustrate its application to several plant materials.
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http://dx.doi.org/10.1007/978-1-4939-3393-8_13DOI Listing
November 2016

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

New Phytol 2015 Jan 25;205(2):653-65. Epub 2014 Sep 25.

Department of Ornamental Horticulture, Agriculture Research Organization, The Volcani Center, PO Box 6, Beit Dagan, 50250, Israel.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.
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http://dx.doi.org/10.1111/nph.13038DOI Listing
January 2015

Maternal C677T MTHFR polymorphism and environmental factors are associated with cleft lip and palate in a Mexican population.

J Investig Med 2013 Aug;61(6):1030-5

Laboratory of Toxicology and Environmental Health, Academic Unit of Chemical-Biological Sciences, Autonomous University of Guerrero, Chilpancingo, Gro, Mexico.

Introduction: Nonsyndromic cleft lip and cleft palate (CL/P) is associated with environmental, nutritional, and genetic factors. Maternal polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene have been associated with CL/P.

Objectives: To determine the relationship between the risk of having a child with CL/P and maternal C677T and A1298C MTHFR polymorphisms, the intake of folate supplements, and exposure to environmental factors during the first trimester of pregnancy, a case-control study of Mexican mothers (88 case mothers and 116 control mothers) was conducted.

Methods: A questionnaire was used to assess exposure to environmental factors. The C677T and A1298C polymorphisms were identified by polymerase chain reaction with restriction fragment length polymorphism.

Results: Mothers with the 677CT or 677TT genotype had a higher risk of having a child with CL/P than mothers with the 677CC genotype (odds ratio [OR], 2.4; 95% confidence interval [CI], 1.1-5.7). An increased risk of having a child with CL/P was associated with the lack of folate supplementation during the first trimester of pregnancy (OR, 3.8; 95% CI, 1.9-7.6), and this risk was greater in the mothers with the 677TT or 677CT genotype than mothers who reported taking folate supplements and had the 677CC genotype (OR, 11.2; 95% CI, 3.3-37.5). Pesticide exposure was associated with CL/P. There was no significant association between either the A1298C variant or tobacco exposure and the risk of CL/P.

Conclusion: These results suggest that gene-environment interactions play an important role in the development of CL/P.
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http://dx.doi.org/10.2310/JIM.0b013e31829a7e7eDOI Listing
August 2013

Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

Plant Physiol 2013 Jul 17;162(3):1486-96. Epub 2013 May 17.

Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.

Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.
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http://dx.doi.org/10.1104/pp.113.220558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707533PMC
July 2013

Evaluation of synthetic acridones and 4-quinolinones as potent inhibitors of cathepsins L and V.

Eur J Med Chem 2012 Aug 21;54:10-21. Epub 2012 Apr 21.

Departamento de Química, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil.

Cathepsins, also known as lysosomal cysteine peptidases, are members of the papain-like peptidase family, involved in different physiological processes. In addition, cathepsins are implicated in many pathological conditions. This report describes the synthesis and evaluation of a series of N-arylanthranilic acids, acridones, and 4-quinolinones as inhibitors of cathepsins V and L. The kinetics revealed that compounds of the classes of acridones are reversible competitive inhibitors of the target enzyme with affinities in the low micromolar range. They represent promising lead candidates for the discovery of novel competitive cathepsin inhibitors with enhanced selectivity and potency. On the other hand, 4-quinolinones were noncompetitive inhibitors and N-arylanthranilic acids were uncompetitive inhibitors.
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http://dx.doi.org/10.1016/j.ejmech.2012.04.002DOI Listing
August 2012

Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don.

AoB Plants 2012 2;2012:pls002. Epub 2012 Mar 2.

IBMC - Instituto de Biologia Molecular e Celular , Universidade do Porto , Rua do Campo Alegre, 823, 4150-180 Porto , Portugal.

Background And Aims: Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size.

Methodology: The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology.

Principal Results: The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp.

Conclusions: The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.
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http://dx.doi.org/10.1093/aobpla/pls002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292738PMC
August 2012

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

J Exp Bot 2011 May 28;62(8):2841-54. Epub 2011 Feb 28.

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), PO Box 164, E-30100 Campus University Espinardo (Murcia), Spain.

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.
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http://dx.doi.org/10.1093/jxb/erq458DOI Listing
May 2011

Fusion with fluorescent proteins for subcellular localization of enzymes involved in plant alkaloid biosynthesis.

Methods Mol Biol 2010 ;643:275-90

IBMC - Institute of Cellular and Molecular Biology, Porto University, Porto, Portugal.

To establish the role in alkaloid metabolism of candidate genes identified in silico or by Omics approaches, it may be essential to determine the subcellular localization of the encoded proteins. The fusion with fluorescent proteins (FP) may now be used as a quite effective and reliable tool to investigate this question. The methodology involves the choice of the FP, the design and production of the appropriate FP fusions, and the use of a transient or stable transformation protocol applied to a homologous or heterologous plant system. This chapter describes the application of this methodology to an enzyme involved in indole alkaloid biosynthesis, with general considerations on the development of the approach.
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http://dx.doi.org/10.1007/978-1-60761-723-5_19DOI Listing
September 2010

A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus: Can peroxidases, secondary metabolites and arabinogalactan proteins be partners in microcompartmentation of cellular reactions?

Plant Signal Behav 2008 Oct;3(10):899-901

IBMC-Instituto de Biologia Molecular e Celular; Universidade do Porto; Porto Portugal.

Plants possess a unique metabolic diversity commonly designated as secondary metabolism, of which the anticancer alkaloids from Catharanthus roseus are among the most studied. Recently, in a classical function-to-protein-to-gene approach, we have characterized the main class III peroxidase (Prx) expressed in C. roseus leaves, CrPrx1, implicated in a key biosynthetic step of the anticancer alkaloids. We have shown the vacuolar sorting determination of CrPrx1 using GFP fusions and we have obtained further evidence supporting the role of this enzyme in alkaloid biosynthesis, indicating the potential of CrPrx1 as a molecular tool for the manipulation of alkaloid metabolism. Here, we discuss how plant cells may regulate Prx reactions. In fact, Prxs form a large multigenic family whose members accept a broad range of substrates and, in their two subcellular localizations, the cell wall and the vacuole, Prxs co-locate with a large variety of secondary metabolites which can be accepted as substrates. How then, are Prx reactions regulated? Localization data obtained in our lab suggest that arabinogalactan proteins (AGPs) and Prxs may be associated in membrane microdomains, evocative of lipid rafts. Whether plasma membrane and/or tonoplast microcompartmentation involve AGPs and Prxs and whether this enables metabolic channeling determining Prx substrate selection are challenging questions ahead.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634410PMC
http://dx.doi.org/10.4161/psb.3.10.6576DOI Listing
October 2008

Conjugated linoleic acid-induced toxic hepatitis: first case report.

Dig Dis Sci 2009 May 23;54(5):1141-3. Epub 2008 Aug 23.

Gastroenterology Department, Covilhã University Hospital, Quinta do Alvito, 6000-251 Covilhã, Portugal.

A 46-year-old female patient was referred to our department with presenting symptoms of asthenia, jaundice, and pruritus. There was no medical history or clinical evidence of viral hepatitis, autoimmune hepatitis, hemochromatosis, or Wilson's disease. The patient revealed that 14 days prior to admission she had begun self-medicating with conjugated linoleic acid (CLA) to reduce body fat, leading to the suspicion of CLA hepatotoxicity, which was subsequently confirmed by a liver biopsy. After the patient ceased to ingest CLA, liver enzymes levels normalized. To the best of our knowledge, this is the first report of hepatotoxicity due to CLA ingestion.
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http://dx.doi.org/10.1007/s10620-008-0461-1DOI Listing
May 2009

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway.

Planta 2008 May 14;227(6):1255-68. Epub 2008 Feb 14.

Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, Porto, Portugal.

Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2(a )mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells.
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http://dx.doi.org/10.1007/s00425-008-0697-1DOI Listing
May 2008

Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus.

Plant Physiol 2008 Feb 7;146(2):403-17. Epub 2007 Dec 7.

John Innes Centre, Norwich NR4 7UH, United Kingdom.

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.
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http://dx.doi.org/10.1104/pp.107.107060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245823PMC
February 2008

Nanoleakage phenomenon on deproteinized human dentin.

J Appl Oral Sci 2007 Aug;15(4):285-91

Department of Restorative Dentistry, School of Dentistry, Federal Fluminense University, Ingá, Niterói, RJ, Brazil.

Objective: The purpose of this study was to evaluate the influence of dentin deproteinization on the nanoleakage phenomenon.

Material And Methods: Class V cavities were prepared in 12 human molars with cervical margins located in dentin. The cavities were assigned to 2 groups (n=6) according to dentin treatment: Group I - dentin treated in accordance with the manufacturer's instructions and Group II - dentin treated following the manufacturer's instructions + 10% NaOCl. Each group was sub-divided into three groups, according to the DBS (dentin bonding system) used: Scotchbond Multi Purpose (SBMP), Prime & Bond NT (PB) and Clearfil SE Bond (SE), which were applied according to manufacturer's instructions. The cavities were restored with composite resin, and the specimens were immersed in a tracer agent (AgNO3 50%) for 24 h. The teeth were sectioned buccolingually through the center of the restorations, and nanoleakage pattern was evaluated by scanning electron microscopy (SEM) using the backscattered electron image mode.

Results: SEM analysis showed different nanoleakage patterns for each DBS. Irrespective of dentin treatments, all SBMP specimens showed nanoleakage. SE did not show any nanoleakage with both dentin treatments used. PB showed nanoleakage within the hybrid layer only in Group I.

Conclusions: The influence of dentin deproteinization on the nanoleakage phenomenon was dependent on dentin bonding system formulation and bonding strategies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327431PMC
http://dx.doi.org/10.1590/s1678-77572007000400009DOI Listing
August 2007

Nanoleakage and microshear bond strength in deproteinized human dentin.

J Biomed Mater Res B Appl Biomater 2007 May;81(2):336-42

Department of Restorative Dentistry, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.

This study evaluated the influence of dentin deproteinization with NaOCl on the microshear bond strength (microSBS) and the nanoleakage patterns of three dentin bonding systems (DBS). Occlusal dentin surfaces, obtained from extracted noncarious human molars, were divided into two experimental groups, according to dentin surface treatment: Group I-37% H(3)PO(4)/15s and Group II-37% H(3)PO(4)/15s + 10% NaOCl/1 min. The dentin surfaces were bonded with one of the following DBS: Scotchbond Multipurpose-SBMP, Prime & Bond NT-PB and Clearfil SE Bond-SE. After 1 week storage in water at 37 degrees C, the specimens were subjected to the microSBS test. The data were analyzed by two-way ANOVA and Student-Newman-Keuls' test (p = 0.05). The nanoleakage was evaluated using scanning electron microscopy (SEM) in backscattered electron imaging regime. No significant difference in microSBS between dentin treatments was found for SBMP. For PB, microSBS increased after NaOCl dentin treatment. SE showed a reduction in microSBS in deproteinized specimens. SEM analysis showed different nanoleakage patterns for each DBS. Irrespective of dentin treatments, all SBMP specimens showed nanoleakage. SE did not show nanoleakage with the two dentin treatments. PB showed nanoleakage within the hybrid layer only in acid-etched specimens. The influence of dentin deproteinization was dependent on the dentin bonding system formulation.
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http://dx.doi.org/10.1002/jbm.b.30670DOI Listing
May 2007

[Differential effects on serum IGF-1 of tibolone (5 mg/day) vs combined continuous estrogen/progestagen in post menopausal women].

Rev Med Chil 2003 Oct;131(10):1151-6

Departamentos de Obstetricia y Ginecología y de Medicina, Facultad de Medicina (Campus Oriente), Universidad de Chile, Santiago, Chile.

Background: Tibolone has estrogenic, androgenic and progestational effects and is used in post menopausal women. It apparently has weaker effects on endometrial proliferation and mammary stimulation than conventional hormone replacement therapy.

Aim: To compare the metabolic effects of tibolone (5 mg/day) and continuous combined conjugated estrogens/medroxyprogesterone acetate in postmenopausal women.

Patients And Methods: Postmenopausal women, aged 45 to 60 years old, receiving estradiol valerate and medroxyprogesterone were included in the study. After a two months wash out period, in a double blind fashion, they were randomly assigned to oral tibolone 5 mg/day or equine conjugated estrogens 0.625 mg + medroxiprogesterone acetate 2.5 mg/day (ECE/MPA). At baseline, 30 and 45 days of treatment, fasting serum osteocalcin, somatomedin C (IGF-1, insulin-like growth factor 1), growth hormone (GH), and follicle stimulating hormone and first morning urine calcium and creatinine were measured.

Results: Thirty women were studied. There was more than 50% fall in urine calcium with either tibolone or ECE/MPA, while fasting GH or osteocalcin did not show significant changes. Serum IGF-1 increased significantly with tibolone at basal, 30 (+109%) and 45 days of treatment and did not change in the ECE/MPA group.

Conclusions: Tibolone (5 mg/day) and ECE/MPA induced a similar reduction in urinary calcium. Tibolone increased serum IGF-1 levels. This may be due to undetected increment of overall GH secretion or to a specific action or IGF-1 generation from the liver and appears to be a novel differential effect of tibolone.
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October 2003
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