Determination of the secondary structure of Kluyveromyces lactis beta-galactosidase by circular dichroism and its structure-activity relationship as a function of the pH

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.     

Integrating stakeholders’ demands and scientific knowledge on ecosystem services in landscape planning

The conflict between conservation and timber production is shifting in regions such as Biscay (Basque Country, northern Spain) where planted forests are no longer profitable without public subsidies and environmentalist claim that public subsidies should be reoriented to the regeneration of natural forest. This paper develops an approach that integrates scientific knowledge and stakeholders’ demands to provide decision-making guidelines for the development of new landscape planning strategies while considering ecosystem services. First, a participatory process was conducted to develop a community vision for the region’s sustainable future considering the opportunities and constrains provided by the landscape and its ecosystems. In the participatory process forest management was considered an important driver for the region`s landscape development and forest multi-functionality was envisioned as a feasible attractive alternative. The participatory process identified a knowledge gap on the synergies and trade-offs between biodiversity and carbon storage and how these depend on different forest types. Second, to study the existing synergies and trade-offs between biodiversity and carbon storage and disentangle the identified knowledge gap, a GIS-based research was conducted based on spatially explicit indicators. Our spatial analysis results showed that natural forests’ contribution to biodiversity and carbon storage is higher than that of the plantations with exotic species in the region. The results from the spatial analysis converged with those from the participatory process in the suitability of promoting, where possible and appropriate, natural forest ecosystems restoration. This iterative learning and decision making process is already showing its effectiveness for decision making, with concrete examples of how the results obtained with the applied approach are being included in planning and decision-making processes.     

Effects of Fumonisin B1-Containing Feed on the Muscle Proteins and Ice-Storage Life of White Shrimp (Litopenaeus vannamei)

Juvenile (5.5–5.7 g) white leg shrimp were exposed to mycotoxin in one indoor trial by feeding fumonisin B₁ (FB₁) at levels from 0.2 to 2.0 μg g^?1 FB₁ for 30 days. Shrimp growth was affected after exposure to more than 0.6 μg g^?1 FB₁. Soluble muscle protein concentration decreased, and changes in myosin thermodynamic properties were observed in shrimp after 30 days of exposure to FB₁. Marked histological changes in tissue of shrimp fed a diet containing FB₁ at 2.0 μg g^?1 were also observed. Shrimp fed diets containing more than 0.6 μg g^?1 FB₁ showed greater decrease in shear forces after 12 days of ice storage.     

Evaluating potassium phosphonate injections for the control of <Emphasis Type="Italic">Quercus ilex</Emphasis> decline in SW Spain: implications of low soil contamination by <Emphasis Type="Italic">Phytophthora cinnamomi</Emphasis> and low soil water content on the effectiveness of treatments

The Iberian forests are suffering severe disease and mortality as a result of decline, with Quercus ilex the major species at risk. Trunk injections with potassium phosphonate, which have been used successfully to control Phytophthora cinnamomi, were tested against decline. In an area in which P. cinnamomi was isolated, Q. ilex trees showing different degrees of decline were trunk-injected. Soil properties, and measurements of soil water content (θ) and depth to soil water table were assessed at three sites with markedly different decline incidences. Over the 5 years following the initiation of the experiment, mean symptoms among spring-treated trees and autumn-treated trees, or among trees injected twice a year (spring and autumn), once a year, and non-injected, were not significantly different. No effects of the treatments on shoot growth and acorn production were observed. However, θ values under trees which recovered from decline were higher than θ values under trees which did not recover from decline. At the site with the highest incidence of decline and tree mortality, P. cinnamomi was rarely isolated, and the presence of gravel, soil infiltration capacities and water table depth values were significantly higher than at the other sites, water stress being more likely to contribute to decline than P. cinnamomi. In areas in which θ is low, the distribution of phosphonate on the tree would be limited. Since the thresholds for phytotoxicity of potassium phosphonate in Q. ilex trees at the site studied would be higher than the amounts used, rates of the chemical slightly less than those that cause phytotoxicity should be tested.     

A wide diversity of zoonotic intestinal parasites infects urban and rural dogs in Neuquén,Patagonia, Argentina

The presence of parasites was investigated by the examination of 1944 dog faecal samples collected from urban (n = 646) and rural (n = 1298) areas of the province of Neuquén, Patagonia, Argentina. Parasitic agents (PA) were found in 37.86% of samples. A total of 15 different PA were detected, including Toxocara canis (16.35%), Taenia spp./Echinococcus spp. (12.65%), Trichurisvulpis (6.06%), Giardia spp. (1.29%), Toxascaris leonina (0.56%), Ancylostomacaninum (0.41%), Dipylidium caninum (0.31%), Diphyllobothrium spp. (0.10%), among others. Several of these PA are recognized as zoonotic agents. Therefore, the results of this investigation revealed that local population is exposed to a broad spectrum of zoonotic parasites by means of environmental contamination with dog faeces. Prevalence of PA was slightly higher in rural (40.06%) than in urban (33.44%) locations. Distribution of groups of PA (cestodes, nematodes, and protozoa) showed statistical differences between both habitats. Prevalence of cestodes (18.18%) and protozoa (11.86%) was significantly higher in the rural environment than in urban areas and nematodes (29.10%) were more frequent in urban locations. Infection of dogs with Linguatula serrata and Cryptosporidium sp. was demonstrated for the first time in Neuquén. Rural dogs of the study area are under hydatic disease control program, which includes treatment with praziquantel every 6 weeks; thus, the finding of high level of cestode infection in these areas is of great relevance. The epidemiology of zoonotic parasitic infections in urban and rural dogs showed different patterns and, in consequence, different control measurements should be applied in each location.     

Impact of glyphosate application on microbial biomass and metabolic activity in a Vertic Argiudoll from Argentina

The widespread use of genetically modified crops tolerant to glyphosate, has strongly promoted the use of this herbicide. This work evaluates the effect of increasing doses of glyphosate on biomass and metabolic activity and metabolic quotient of soil microbiota under controlled conditions. Commercial formulation of glyphosate (48%) was sprayed over a Vertic Argiudoll placed in trays, at doses of 0.48, 0.96, 1.92 and 3.84 L a.i ha^?1. Doses usually applied in the field are between 0.96 and 1.92 L a.i ha^?1. Treatments and control were incubated at 25 °C and 75% of water holding capacity. After 4 and 45 days from glyphosate application, carbon from microbial biomass (C-MB), microbial respiration rate (MR), metabolic quotient (qCO₂), and dehydrogenase activity (DA) were determined. The C-MB at 4 days of incubation did not differ (P < 0.05) between the control and the doses of 0.48 and 0.96 L, while it was significantly lower in the highest doses. After 45 days, C-BM in the dose of 3.84 L was significantly lower than the control. The MR showed significant differences over the time but not between doses. Significant differences were found in qCO₂ between doses in both periods of incubation. Dehydrogenase activity was significantly higher in the treatments with glyphosate than in the control at the beginning of incubation. Treatment with 0.48 L presented the highest value after 45 days, while the dose of 3.84 L had lowest DA. The variables analyzed showed an initial inhibitory effect that affected the microbial cells. However, this effect was temporary at doses equivalent or higher than those usually applied in the field.     

Role of lysine epsilon-amino groups of beta-lactoglobulin on its activating effect of Kluyveromyces lactis beta-galactosidase

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.     

Water distribution in brine salted cod (Gadus morhua) and salmon (Salmo salar): a low-field 1H NMR study

Low-field (LF) (1)H NMR T 2 relaxation measurements were used to study changes in water distribution in lean (Atlantic cod) and fatty (Atlantic salmon) fish during salting in 15% NaCl and 25% NaCl brines. The NMR data were treated by PCA, continuous distribution analysis, and biexponential fitting and compared with physicochemical data. Two main water pools were observed in unsalted fish, T 21, with relaxation times in the range 20-100 ms, and T 22, with relaxation times in the range 100-300 ms. Pronounced changes in T 2 relaxation data were observed during salting, revealing changes in the water properties. Salting in 15% brine lead to a shift toward longer relaxation times, reflecting increased water mobility, whereas, salting in saturated brines had the opposite effect. Water mobility changes were observed earlier in the salting process for cod compared to salmon. Good linear correlations ( F 相似文献