Browsing by Issue Date, starting with "2012-11-08"
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- Functional activity of seaweed extracts from the north portuguese coastPublication . Mendes, Marta Sofia de Almeida; Gomes, Ana Maria Pereira; Carvalho, Ana Paula Santos deA utilização de algas marinhas como fontes potenciais de compostos nutracêuticos e farmacêuticos tem aumentado recentemente devido à constatação de que estas contêm compostos bioactivos, com actividades antioxidante e antimicrobiana (entre outras actividades), que podem inibir o crescimento de algumas bactérias contaminantes e/ou patogénicas e de leveduras prevenindo a deterioração de alimentos ou a infecção ou contribuindo mesmo para o seu melhor controlo. O litoral português alberga uma grande biodiversidade no que concerne a algas marinhas, porém muitas encontram-se por caracterizar em termos de propriedades funcionais. Neste contexto, o objectivo deste trabalho foi estabelecer um procedimento melhorado para a obtenção de extractos de algas marinhas e testar a sua actividade antimicrobiana contra espécies selecionadas de leveduras, bactérias Gram positivas e Gram negativas, bem como a sua actividade antioxidante. Para tentativamente atestar o seu comportamento, os perfis lipídico e fenólico foram testados. As algas utilizadas neste estudo, incluindo as de aquacultura integrada e as de habitat natural, foram obtidas no Norte de Portugal. A alga Gracilaria vermiculophylla foi usada para os ensaios de optimização do processo de extracção, enquanto as Gracilaria vermiculophylla, Porphyra dioica e Chondrus crispus foram utilizadas para os ensaios de actividade antimicrobiana e antioxidante. Os estudos de optimização centraram-se na definição dos pré-tratamentos (secagem) e da temperatura a utilizar durante o processo de extracção. Os resultados revelaram que os organismos testados foram mais sensíveis aos extractos obtidos com algas secas, continuamente processados a temperaturas mais elevadas. Posteriormente, extratos obtidos com três diferentes solventes (acetato de etilo, éter dietílico e metanol:água) foram testados. No que diz respeito à avaliação da actividade antimicrobiana, as espécies testadas incluíram (i) bactérias Gram negativas - Escherichia coli, Salmonella enteritidis e Pseudomonas aeruginosa; (ii) bactérias Gram positivas – Listeria innocua, Bacillus cereus, Enterococcus faecalis, Lactobacillus brevis, Staphylococcus aureus, todas de origem alimentar, e uma estirpe de Staphylococcus aureus de origem clínica, e (iii) a levedura Candida spp. também de origem clinica. Os testes para avaliar a actividade antimicrobiana dos extractos foram realizados utilizando o método de difusão em agar e os resultados indicaram uma forte actividade antimicrobiana dos extractos de acetato de etilo, quando comparado com os extractos de metanol e éter dietílico e mostraram uma tendência fraca para a inibição de microrganismos Gram positivos. O perfil de ácidos gordos de extractos de acetato de etilo revelou uma predominância de ácidos gordos saturados (SFA), especialmente o acido palmítico (16:0), seguido por ácidos gordos polinsaturados (PUFA) e ácidos gordos monoinsaturados (MUFA) e mostrou um teor mais elevado de ácidos gordos em G. vermiculophylla e P. dioica de aquacultura. Tendo em conta os resultados obtidos para a actividade antioxidante, foi demonstrado que os extractos metanólicos apresentaram actividade mais elevada quando comparada com os outros solventes testados. O perfil fenólico revelou que os extractos metanólicos mostraram quantidades mais elevadas de compostos fenólicos, tais como catequinas e ácido protocatecuico, o que pode indiciar o seu papel na actividade antioxidante.
- Identification and functional analysis of nematode resistance genesPublication . Silva, Ana Isabel Paulino da; Vasconcelos, Marta Wilton; Monsanto, MiguelPine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). High throughput sequencing allowed the identification of several candidate genes that may be involved in the response to the PWN. With regards to the gene function most commonly identified, the majority of the sequence functions were associated with protein metabolism and carbohydrate metabolism. However, a significant fraction of sequences associated with RNA metabolism were also highly represented. The sequences that were more commonly found in Pinus pinaster were transcription repressors and a translation machinery component: aminoacyl-tRNA synthetase. The cellulose synthase is also important in the disease response, as this gene was up-regulated in infested Pinus pinaster. KEGG analysis revealed that the pathway more commonly found in this study were the pentose pathway, the pathway for glucuronate interconversion, the pathway for phenylanine metabolism, amino acid, sugar and nucleotide metabolism, phenylppropanoid biosynthesis, methane metabolism, and citrate cycle (TCA cycle).
- Controlled release of tethered peptides from alginate hydrogels via enzymatic linker degradationPublication . Barbosa, Mariana Moreira da Silva Alves; Barrias, Cristina Carvalho; Gomes, Paula A. C.Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots and provide their sustained delivery for enhanced effectiveness. The aim of this study was to develop a MMP-responsive hydrogel-based delivery vehicle for the active fragment of the osteogenic growth peptide (OGP10-14, YGFGG), to be used in bone regeneration therapies. For this purpose two alternative peptide sequences were designed. These were synthesized using a solid-phase strategy (SPPS), by extending the original YGFGG sequence at its amine terminus with either a MMP-sensitive (PVGLIG) or a MMP-insensitive (scrambled, GIVGPL) domain. The designed sequences were then chemically conjugated to alginate, a hydrogel-forming polymer. The hypothesis behind the study was that in presence of specific MMPs (namely MMP-2) the PVLIG linker could be enzymatically cleaved leading to the release of OGP10-14 from the alginate hydrogel vehicle. In contrast, OGP would remain grafted when using the MMP-insensitive (scrambled) domain as linker. The oligopeptide sequences were successfully synthesized by SPPS, and analyzed by liquid chromatography-mass spectrometry (LC-MS). Both were subsequently characterized in terms of their susceptibility to proteases. The extent of peptide cleavage upon incubation with MMP-2 was determined using the fluorescamine assay, and changes in the peptides molar mass were determined by LC-MS. The PVGLIG sequence was effectively hydrolyzed by MMP-2 at the predicted cleavage site (G↓L). The scrambled sequence was also partially cleaved, albeit unspecifically and at much lower rates. The kinetics of peptide digestion was further analyzed using custom-made FRET sequences, by monitoring the temporal increase in fluorescence resulting from peptide cleavage. Both peptides were partially cleaved by proteases present in serum, but the PVGLIG peptide was much more responsive to MMP-2 than the scrambled peptide. Preliminary studies were carried out to evaluate the bioactivity of OGP10-14 when presented to human mesenchymal stem cells (hMSC) in both peptide sequences. Osteogenic differentiation was more efficiently promoted when OGP was incorporated in the MMP-sensitive oligopeptide. Both peptides were conjugated to alginate via carbodiimide-mediated chemical grafting. The amount of immobilized peptide was quantified by UV-Vis and by the Total Protein Biccinchoninic acid (BCA) assay, which demonstrated that the coupling procedure was successful, with an efficiency around 41% (PVGLIG) to 62% (scrambled). MMP-2 digestion of peptide-alginate conjugates was analyzed by the fluorescamine assay. Higher enzymatic cleavage was observed in peptides-alginate conjugates with the MMP-sensitive PVGLIG linker. Overall, this study provided proof-of-concept on the correct design of alginate-PVGLIG-conjugates, in the sense that, as expected, these were sensitive to MMP-2 mediated cleavage and may be a useful platform for the in situ delivery of OGP.