Purification and identification of cutinases from <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis> and <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.     

Oxygen distribution in the fluid/gel phases of lipid membranes

The interactions between oxygen and lipid membranes play fundamental roles in basic biological processes (e.g., cellular respiration). Obviously, membrane oxidation is expected to be critically dependent on the distribution and concentration of oxygen in the membrane. Here, we combined theoretical and experimental methods to investigate oxygen partition and distribution in lipid membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a temperature range between 298 and 323 K, specifically focusing on the changes caused by the lipid phase and phase transition. Even though oxygen is known to be more concentrated in the center of fluid phase membranes than on the headgroup regions, the distribution profile of oxygen inside gel-phase bilayers remained to be determined. Molecular dynamics simulations now show that the distribution of oxygen inside DPPC bilayers dramatically changes upon crossing the main transition temperature, with oxygen being nearly depleted halfway from the headgroups to the membrane center below the transition temperature. In a parallel approach, singlet oxygen luminescence emission measurements employing the photosensitizer Pheophorbide-a (Pheo) confirmed the differences in oxygen distribution and concentration profiles between gel- and fluid-phase membranes, revealing changes in the microenvironment of the embedded photosensitizer. Our results also reveal that excited triplet state lifetime, as it can be determined from the singlet oxygen luminescence kinetics, is a useful probe to assess oxygen distribution in lipid membranes with distinct lipid compositions.     

Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.     

Trace Metal Concentration in a Temperate Freshwater Reservoir Seasonally Subjected to Blooms of Toxin-Producing Cyanobacteria

In situ interactions between cyanobacteria and metals were studied at Torrão reservoir (Tâmega River, North Portugal). The metal content of water and sediments from the reservoir was monitored monthly at Marco de Canaveses (seasonally subjected to toxic blooms of Microcystis aeruginosa) and upstream at Amarante (no blooms recorded), for 16 months. During the 16 months of the study period, M. aeruginosa bloomed twice at Marco de Canaveses, firstly forming a scum, and later with colonies scattered throughout the reservoir. Metals Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn were analysed in the sediment and in the water column. Cu-binding ligands in water were also determined. When no blooms were taking place, average metal levels for water and sediment were not statistically different at both locations. Therefore, it was considered that the absence of cyanobacteria blooms at Amarante was not due to differences in metal content. When blooms were taking place at Marco de Canaveses, a significant increase of metal levels in the sediment occurred simultaneously. Sediment quality guidelines showed that during this period, Cu and Pb concentrations (32.3 and 43.2 mg kg^?1, respectively) were potentially toxic. However, quantification of the exchangeable metal fraction indicated that these metals were probably not bioavailable. Concentration of Cu-binding ligands in water was higher during the blooms, indicating that cyanobacteria are capable of changing the metal speciation in situ in a reservoir.     

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Developmental and physiological challenges of octopus (Octopus vulgaris) early life stages under ocean warming

The ability to understand and predict the effects of ocean warming (under realistic scenarios) on marine biota is of paramount importance, especially at the most vulnerable early life stages. Here we investigated the impact of predicted environmental warming (+3 °C) on the development, metabolism, heat shock response and antioxidant defense mechanisms of the early stages of the common octopus, Octopus vulgaris. As expected, warming shortened embryonic developmental time by 13 days, from 38 days at 18 °C to 25 days at 21 °C. Concomitantly, survival decreased significantly (~29.9 %). Size at hatching varied inversely with temperature, and the percentage of smaller premature paralarvae increased drastically, from 0 % at 18 °C to 17.8 % at 21 °C. The metabolic costs of the transition from an encapsulated embryo to a free planktonic form increased significantly with warming, and HSP70 concentrations and glutathione S-transferase activity levels were significantly magnified from late embryonic to paralarval stages. Yet, despite the presence of effective antioxidant defense mechanisms, ocean warming led to an augmentation of malondialdehyde levels (an indicative of enhanced ROS action), a process considered to be one of the most frequent cellular injury mechanisms. Thus, the present study provides clues about how the magnitude and rate of ocean warming will challenge the buffering capacities of octopus embryos and hatchlings’ physiology. The prediction and understanding of the biochemical and physiological responses to warmer temperatures (under realistic scenarios) is crucial for the management of highly commercial and ecologically important species, such as O. vulgaris.     

Early-life exposure to climate change impairs tropical shark survival

Sharks are one of the most threatened groups of marine animals worldwide, mostly owing to overfishing and habitat degradation/loss. Although these cartilaginous fish have evolved to fill many ecological niches across a wide range of habitats, they have limited capability to rapidly adapt to human-induced changes in their environments. Contrary to global warming, ocean acidification was not considered as a direct climate-related threat to sharks. Here we show, for the first time, that an early ontogenetic acclimation process of a tropical shark (Chiloscyllium punctatum) to the projected scenarios of ocean acidification (ΔpH = 0.5) and warming (+4°C; 30°C) for 2100 elicited significant impairments on juvenile shark condition and survival. The mortality of shark embryos at the present-day thermal scenarios was 0% both at normocapnic and hypercapnic conditions. Yet routine metabolic rates (RMRs) were significantly affected by temperature, pH and embryonic stage. Immediately after hatching, the Fulton condition of juvenile bamboo sharks was significantly different in individuals that experienced future warming and hypercapnia; 30 days after hatching, survival rapidly declined in individuals experiencing both ocean warming and acidification (up to 44%). The RMR of juvenile sharks was also significantly affected by temperature and pH. The impact of low pH on ventilation rates was significant only under the higher thermal scenario. This study highlights the need of experimental-based risk assessments of sharks to climate change. In other words, it is critical to directly assess risk and vulnerability of sharks to ocean acidification and warming, and such effort can ultimately help managers and policy-makers to take proactive measures targeting most endangered species.     

Prevalence of Overweight,Obesity, and Abdominal Obesity in a Representative Sample of Portuguese Adults

This study determined the prevalence of overweight, obesity, and abdominal obesity in the Portuguese adults and examined the relationship between above mentioned prevalences and educational level. Body mass, stature, and waist circumference were measured in a representative sample of the Portuguese population aged 18–103 years (n = 9,447; 18–64 years: n = 6,908; ≥65 years: n = 2,539). Overweight and obesity corresponded to a body mass index ranging between 25–29.9 kg/m² and ≥30 kg/m², respectively. Abdominal obesity was assessed as >102 cm for males and >88 cm for females. After adjusting for educational level, the combined prevalences of overweight and obesity were 66.6% in males and 57.9% in females (18–64 years). Respective values in older adults (≥65 years) were 70.4% for males and 74.7% for females. About 19.3% of adult males and 37.9% of adult females presented abdominal obesity. Correspondent values in older adults were 32.1%, for males, and 69.7%, for females. In adults, low educational level was related to an increased risk for overweight (OR = 2.54; 95% CI: 2.08–3.09), obesity (OR = 2.76; 95% CI: 2.20–3.45), and abdominal obesity (OR = 5.48; 95% CI: 4.60–6.52). This reinforces the importance of adjusting public health strategies for educational level.