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- Biological characterization of Cynara cardunculus L. Methanolic extracts: antioxidant, anti-proliferative, anti-migratory and anti-angiogenic activitiesPublication . Velez, Zélia; Campinho, Marco António; Guerra, Ângela R.; García, Laura; Ramos, Patricia; Guerreiro, Olinda; Felício, Laura; Schmitt, Fernando; Duarte, MariaCynara cardunculus (Cc) is a multipurpose species; beyond its use in southwestern European cuisine, it is also used for the production of solid biofuel, seed oil, biodiesel, paper pulp and cheese, as well as animal feed. In addition, Cc has a long tradition of use in folk medicine as a diuretic and liver protector. The value of this species as a source of bioactive compounds is known; however, pharmacological use would further increase its cultivation. The main goal of the current work was to evaluate the potential of Cc as source of anti-carcinogenic phytochemicals. Different methanolic extracts obtained from wild and cultivated plants were tested for antioxidant activity and effect on breast tumor cell viability. The most effective extract, both as antioxidant and inhibition of tumor cell viability, was tested for effects on angiogenesis and tumor cell migration capacity. All the extracts tested had high antioxidant activity; however, only green leaves and dry head extracts exhibit anti-proliferative activity. Green cultivated leaves (GCL) were the most effective extract both as antioxidant and inhibiting the proliferation of tumor cells; it is equally active inhibiting tumor cell migration and in vivo angiogenesis. GCL extract is an effective inhibitor of several key points in tumor development and thus a promising source of anti-carcinogenic phytochemicals.
- Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax)Publication . Pinto, P.; Velez, Zélia; Sousa, Carmen; Santos, Soraia; Andrade, André; Alvarado, M. V.; Felip, A.; Zanuy, S.; Canario, A.V.M.The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29 Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29 Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates. (C) 2017 Elsevier Inc. All rights reserved.
- Evidence that 1-methyl-l-tryptophan is a food-related odorant for the Senegalese sole (Solea senegalensis)Publication . Velez, Zélia; Hubbard, Peter; Hardege, Jörg D.; Welham, K.; Barata, E. N.; Canario, Adelino V. M.Nocturnal animals often rely heavily on olfactory cues to locate their food. This is especially true for fish whose prey live buried in the substrate. The aim of the current study was to identify non-polar odorants released by the ragworm (Hediste diversicolor) that are detected by the olfactory system of the Senegalese sole (Solea senegalensis) and therefore may be food-related cues. Ragworm-conditioned water was passed through a C18 solid-phase extraction cartridge (to remove polar compounds) and the eluate fractionated by reverse phase high-performance liquid chromatography (HPLC). The fractions were then tested for olfactory activity in the sole by the electro-olfactogram (EOG). Most olfactory activity was found in the first two fractions to elute from the HPLC column (0–4 min). Liquid chromatography-mass spectrometry (LC/MS) of both fractions revealed a base peak with a molecular mass of 219.4 Da and a fragment of 205.3 Da. These data are consistent with a methylated form of tryptophan; synthetic 1-methyl-l-tryptophan had a similar HPLC retention time (2.8 min) and similar LC/MS spectra. Furthermore, sole showed olfactory sensitivity to 1-methyl-l-tryptophan with the lower (left) epithelium being more sensitive than the upper (right). Cross-adaptation (using EOG recording) suggested that the olfactory receptors responding to 1-methyl-l-tryptophan are different in the lower epithelium from the upper. These results suggest that ragworms release 1-methyl-l-tryptophan, or similar tryptophan derivative, and that sole may use this chemical cue to locate and/or identify one of their main prey species. This may help the formation of artificial feeds that are more attractive to sole.
- Olfactory transduction pathways in the Senegalese sole Solea senegalensisPublication . Velez, Zélia; Hubbard, Peter; Barata, E. N.; Canario, Adelino V. M.This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro-olfactogram. Receptor mechanisms were assessed by cross-adaptation with amino acids (L-cysteine, L-phenylalanine and 1-methyl-L-tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1-methyl-L-tryptophan and Lphenylalanine (food-related odorants) in the lower epithelium, and for taurocholic acid (conspecificderived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC-mediated transduction (IC50; 15–55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) (using SQ-22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non-PLC and non-AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non-PLC and non-AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.
- Short- and medium-term exposure to ocean acidification reduces olfactory sensitivity in Gilthead SeabreamPublication . Velez, Zélia; Roggatz, Christina C.; Benoit, David M.; Hardege, Jörg D.; Hubbard, PeterThe effects of ocean acidification on fish are only partially understood. Studies on olfaction are mostly limited to behavioral alterations of coral reef fish; studies on temperate species and/or with economic importance are scarce. The current study evaluated the effects of short- and medium-term exposure to ocean acidification on the olfactory system of gilthead seabream (Spares aurata), and attempted to explain observed differences in sensitivity by changes in the protonation state of amino acid odorants. Short-term exposure to elevated PCO2 decreased olfactory sensitivity to some odorants, such as L-serine, L-leucine, L-arginine, L-glutamate, and conspecific intestinal fluid, but not to others, such as L-glutamine and conspecific bile fluid. Seabream were unable to compensate for high PCO2 levels in the medium term; after 4 weeks exposure to high PCO2 , the olfactory sensitivity remained lower in elevated PCO2 water. The decrease in olfactory sensitivity in high PCO2 water could be partly attributed to changes in the protonation state of the odorants and/or their receptor(s); we illustrate how protonation due to reduced pH causes changes in the charge distribution of odorant molecules, an essential component for ligand-receptor interaction. However, there are other mechanisms involved. At a histological level, the olfactory epithelium contained higher densities of mucus cells in fish kept in high CO2 water, and a shift in pH of the mucus they produced to more neutral. These differences suggest a physiological response of the olfactory epithelium to lower pH and/or high CO2 levels, but an inability to fully counteract the effects of acidification on olfactory sensitivity. Therefore, the current study provides evidence for a direct, medium term, global effect of ocean acidification on olfactory sensitivity in fish, and possibly other marine organisms, and suggests a partial explanatory mechanism.