Nuno M. Pedroso

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.

Modulation of plasma membrane lipid profile and microdomains by H2O2 in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the rate of hydrogen peroxide (H(2)O(2)) diffusion through the plasma membrane decreases during adaptation to H(2)O(2) by a still unknown mechanism. Here, adaptation to H(2)O(2) was observed to modulate rapidly the expression of genes coding for enzymes involved in ergosterol and lipid metabolism. Adaptation to H(2)O(2) also alters plasma membrane lipid composition. The main changes were the following: (a) there was a decrease in oleic acid (30%) and in the ratio between unsaturated and saturated long-chain fatty acids; (b) the phosphatidylcholine:phosphatidylethanolamine ratio increased threefold; (c) sterol levels were unaltered but there was an increased heterogeneity of sterol-rich microdomains and increased ordered domains; (d) the levels of the sterol precursor squalene increased twofold, in agreement with ERG1 gene down-regulation; and (e) C26:0 became the major very long chain fatty acid owing to an 80% decrease in 2-hydroxy-C26:0 levels and a 50% decrease in C20:0 levels, probably related to the down-regulation of fatty acid elongation (FAS1, FEN1, SUR4) and ceramide synthase (LIP1, LAC1) genes. Therefore, H(2)O(2) leads to a reorganization of the plasma membrane microdomains, which may explain the lower permeability to H(2)O(2), and emerges as an important regulator of lipid metabolism and plasma membrane lipid composition.

Use of small and medium-sized water reservoirs by otters in a Mediterranean ecosystem

Water is a limiting factor in Mediterranean regions, being specially important to aquatic species such as the Eurasian otter. The seasonal fluctuation of this resource is often addressed by constructing small and medium-sized water reservoirs. However, their role in the ecology and conservation of Eurasian otters is largely unknown. Our main goals were to assess the level of use of these reservoirs by otters and determine the main factors that may explain the observed levels of use. Intensity of use was determined by signs of otter presence and related to environmental variables using generalized linear models and variation partitioning techniques. Otters were present in the majority of reservoirs, both in the wet and dry seasons. Otter marking intensity was higher during the dry season, and positively associated with abundance of fish and American crayfish, existence of refuges and marking sites, number of watercourses and area of reservoir. In contrast, cattle settlements, annual crops and length of watercourses in the surrounding area negatively affected the use of the reservoirs. Otter diet reflected their opportunistic behaviour through the selection of seasonally available prey and corroborated the importance of American crayfish as a food item. Our results confirm that otters use the majority of small and medium-sized reservoirs in the study area. Despite this, reservoirs may be considered suboptimal habitats and seem to be specially relevant in the dry season, increasing water availability and acting as important feeding areas mainly when close to watercourses with good refuge conditions. Management implications are discussed.

Antimicrobial-Resistant Salmonella Isolated from Eurasian Otters (Lutra lutra Linnaeus, 1758) in Portugal

Eurasian otters (Lutra lutra Linnaeus, 1758) are present in a wide range of aquatic environments. Salmonella isolates have been obtained from otters at rehabilitation centers and in the wild and are sometimes associated with serious illnesses. Antimicrobial-resistant Salmonella were isolated from otter fecal samples collected in March 2006, September 2007, and March 2008 in two river basins in southern Portugal. From 67 samples tested, five were positive for Salmonella (7.58%). None of the isolates were susceptible to all antimicrobials tested, and all were resistant to multiple antimicrobials. Our results confirm the role of otters as potential carriers of Salmonella and the importance of environmental exposure to antimicrobial agents in selection for resistance in bacteria.

The plasma membrane-enriched fraction proteome response during adaptation to hydrogen peroxide in Saccharomyces cerevisiae

Abstract In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H2O2) decreases plasma membrane permeability to H2O2, changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H2O2-induced adaptation effect on the S. cerevisiae plasma membrane-enriched fraction proteome, using two-dimensional gel electrophoresis, that 44 proteins are differentially expressed. Most of these proteins were regulated at a post-transcriptional level. Fourteen of these proteins contain redox-sensitive cysteine residues and nine proteins are associated with lipid and vesicle traffic. In particular, three proteins found in eisosomes and in the eisosome-associated membrane compartment occupied by Can1p were up-regulated (Pil1p, Rfs1p and Pst2p) during adaptation to H2O2. Survival studies after exposure to lethal H2O2 doses using yeast strains bearing a gene deletion corresponding to proteins associated to lipid and vesicle traffic demonstrated for the first time that down-regulation of Kes1p, Vps4p and Ynl010wp and up-regulation of Atp1 and Atp2 increases resistance to H2O2. Moreover, for the pil1Δ strain, H2O2 at low levels produces a hormetic effect by increasing proliferation. In conclusion, these data further confirms the plasma membrane as an active cellular site during adaptation to H2O2 and shows that proteins involved in lipid and vesicle traffic are important mediators of H2O2 adaptation.

O Papel do Programa Lince (LPN/FFI) na Recuperação do Habitat e Presas do Lince-Ibérico no Sul de Portugal

Resumo O lince-iberico Lynx pardinus (Temminck, 1827) e actualmente considerado o felino mais ameacado do mundo. Por esta razao, em 2004, a Liga para a Proteccao da Natureza (LPN) e a Fauna & Flora International (FFI) lancaram o Programa Lince cujo principal objectivo e contribuir para a conservacao e gestao a longo prazo de um corredor de habitats prioritarios para a conservacao do lince-iberico em Portugal. Neste trabalho, sao apresentados alguns dos resultados deste Programa respeitantes a recuperacao das populacoes de coelho-bravo na Serra do Caldeirao e no Sitio Moura/Barrancos. Neste ultimo local o trabalho diz respeito ao Projecto LIFE Lince Moura/Barrancos, co-financiado a 75% pelo Programa LIFE - Natureza da Comissao Europeia (CE). Os principais objectivos definidos foram: i) a realizacao de protocolos de colaboracao com agentes locais; e ii) a recuperacao das populacoes de coelho-bravo. Foram realizados 24 contactos com proprietarios/gestores de caca dos quais resultaram 9 protocolos de colaboracao com planos de gestao para melhorar o habitat e as populacoes de coelho. No âmbito destes protocolos de colaboracao foram realizados 128 abrigos, 160 comedouros e 116 bebedouros em diferentes propriedades/zonas de caca. Apesar de ainda preliminares, os resultados foram bastante positivos. De um modo geral, todas as estruturas implementadas (abrigos, comedouros, bebedouros) foram usadas por coelho-bravo, indicando que estas sao adequadas e utilizadas por esta especie. Tendo em conta os resultados obtidos, espera-se que, cada vez mais, os proprietarios e gestores de caca estejam receptivos para a conservacao da Natureza, nomeadamente do habitat Mediterrânico. Palavras-chave: Coelho-bravo, lince-iberico, gestao, habitat, Programa Lince. Abstract The Iberian lynx Lynx pardinus (Temminck, 1827) is currently considered the most endangered feline in the world. For this reason, in 2004, the Liga para a Proteccao da Natureza (LPN) and Fauna & Flora International (FFI) launched the Lynx Programme whose main objective is to contribute to the conservation and long-term management of a corridor of priority habitats for the conservation of the Iberian lynx in Portugal. Here we present some results of this work relating to the recovery of wild rabbit populations at Serra do Caldeirao and the Moura/Barrancos Site – in this latter place, the work concerns the LIFE Lince Moura/Barrancos project, co-funded in 75% by the European Commission. The main objectives were: i) the establishment of management agreements with local stakeholders, and ii) the recovery of wild rabbit populations. Twenty four contacts were made with landowners/game managers, which resulted in nine management agreements with management artificial shelters, 160 food suppliers and 116 water suppliers were implemented in different estates/hunting areas. Although still preliminary, these results were quite positive. In general, all the implemented structures (artificial shelters, food and water suppliers) were used by wild rabbits, indicating their suitability for this species. Given the results achieved, it is expected that, gradually, landowners and game managers will be more receptive to nature conservation, particularly of this Mediterranean habitat. Keywords : Habitat, Iberian lynx, Lynx Programme, management, wild rabbit.