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Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction
Publication . Gomes, Filipa O.; Maia, Luísa B.; Cordas, Cristina; Moura, Isabel; Delerue-Matos, Cristina; Moura, José J.G.; Morais, Simone
Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors.
Acetylated cashew gum-based nanoparticles for the incorporation of alkaloid epiisopiloturine
Publication . Rodrigues, Jessica do Amaral; Araújo, Alyne Rodrigues de; Pitombeira, Nadia Aline; Plácido, Alexandra; Almeida, Miguel Peixoto de; Veras, Leiz Maria Costa; Delerue-Matos, Cristina; Lima, Filipe Camargo Dalmatti Alves; Neto, Augusto Batagin; Paula, Regina Célia Monteiro de; Feitosa, Judith Pessoa Andrade; Eaton, Peter; Leite, José Roberto Souza A.; Silva, Durcilene Alves da
The natural alkaloid epiisopiloturine has recently become the focus of study for various medicinal properties, particularly for its anti-inflammatory and antischistosomal effect. The incorporation of active molecules in natural polymeric matrices has garnered increasing interest during recent decades. A new derivative of cashew gum successfully obtained by gum acetylation has shown great potential as a carrier in controlled drug release systems. In this work, epiisopiloturine was encapsulated in acetylated cashew gum nanoparticles in order to increase solubility and allow slow release, whereas the morphology results were supported by computer simulations. The particles were produced under a variety of conditions, and thoroughly characterized using light scattering and microscopic techniques. The particles were spherical and highly stable in solution, and showed drug incorporation at high levels, up to 55% efficiency. Using a dialysis-based in vitro assay, these particles were shown to release the drug via a Fickian diffusion mechanism, leading to gradual drug release over approximately 6 h. These nanoparticles show potential for the use as drug delivery system, while studies on their potential anti-inflammatory action, as well as toxicity and efficacy assays would need to be performed in the future to confirm their suitability as drug delivery candidates.
Structure–Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian Cells
Publication . Campelo, Yuri; Ombredane, Alicia; Vasconcelos, Andreanne; Albuquerque, Lucas; Moreira, Daniel; Plácido, Alexandra; Rocha, Jefferson; Fokoue, Harold Hilarion; Yamaguchi, Lydia; Mafud, Ana; Mascarenhas, Yvonne; Delerue-Matos, Cristina; Borges, Tatiana; Joanitti, Graziella; Arcanjo, Daniel; Kato, Massuo; Kuckelhaus, Selma; Silva, Marcos; Moraes, Josué; Leite, José
Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure–function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5⁻10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.
Fluorometric method based on molecular recognition solid-phase extraction for determination of riboflavin in milk and infant formula
Publication . Osório, Marcelo V.; Marques, Sara S.; Oliveira, Hugo M.; Barreiros, Luisa; Segundo, Marcela A.
Riboflavin (vitamin B2) is involved in several biological processes, particularly in energy production, and it is acquired from food ingestion, principally from supplemented food during the first years of life. Therefore, a simple, fast and cost-effective high-throughput method for determination of riboflavin in milk and infant formula is proposed, based on selective extraction using commercially available molecularly imprinted polymers targeted to riboflavin, followed by direct fluorometric determination. Several aspects were studied, namely microplate assay conditions, the composition of eluting solution and the stability of riboflavin in the eluate. Hence, elution using 1% (v/v) acetic acid in methanol or in acetonitrile is recommended, followed by immediate analysis or solvent evaporation, with reconstitution and analysis within 24 h. The proposed method provided a LOD of 0.03 mg L−1, with working range for undiluted samples between 0.125 and 2 mg L−1, and sample throughput of 24 h−1. It was successfully applied to certified reference material NIST-1846 and also to commercial milk and infant formula samples.
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Fundação para a Ciência e a Tecnologia
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5876
Número da atribuição
UID/Multi/04378/2013