A germin-like protein of wheat leaf apoplast inhibits serine proteases

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.     

A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells

The four basic isoforms of the plasma membrane Ca2+ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII(3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca2+ in the native intracellular environment. The level of [Ca2+] in the endoplasmic reticulum and the height of the [Ca2+] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca2+ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.     

Selective excitation of native fluctuations during thermal unfolding simulations: horse heart cytochrome c as a case study

The effect of temperature on the activation of native fluctuation motions during molecular dynamics unfolding simulations of horse heart cytochrome c has been studied. Essential dynamics analysis has been used to analyze the preferred directions of motion along the unfolding trajectories obtained by high temperature simulations. The results of this study have evidenced a clear correlation between the directions of the deformation motions that occur in the first stage of the unfolding process and few specific essential motions characterizing the 300 K dynamics of the protein. In particular, one of those collective motions, involved in the fluctuation of a loop region, is specifically excited in the thermal denaturation process, becoming progressively dominant during the first 500 ps of the unfolding simulations. As further evidence, the essential dynamics sampling performed along this collective motion has shown a tendency of the protein to promptly unfold. According to these results, the mechanism of thermal induced denaturation process involves the selective excitation of one or few specific equilibrium collective motions.     

Bacterial community structure and location in Stilton cheese

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.     

A simple system for monitoring biodiversity in protected areas of a developing country

The achievements of initiatives to strengthen biodiversity conservation in developing countries may be difficult to assess, since most countries have no system for monitoring biodiversity. This paper describes a simple and cost-effective, field-based biodiversity monitoring system developed specifically for areas where specialist staff is lacking. We discuss the preliminary lessons learned from protected areas in the Philippines. Whilst the monitoring system aims to identify trends in biodiversity and its uses so as to guide management action, it also promotes the participation of local people in the management, stimulates discussions about conservation amongst stakeholders and builds the capacity of park staff and communities in management skills. In addition, it seeks to provide people with direction regarding the aims of protected areas, and reinforces the consolidation of existing livelihoods through strengthening community-based resource management systems. The field methods are: (1) standardised recording of routine observations, (2) fixed point photographing, (3) line transect survey, and (4) focus group discussion. Both bio-physical and socio-economic data are used and given equal importance. The system can be sustained using locally available resources. The approach is useful in countries embarking on shared management of park resources with local communities, where rural people depend on use of natural ecosystems, and where the economic resources for park management are limited. We hope this paper will encourage other countries to develop their own biodiversity monitoring system, letting its development become a means for capacity building whilst at the same time supporting the creation of ownership.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Occurrence of an invasive coral in the southwest Atlantic and comparison with a congener suggest potential niche expansion

Tubastraea tagusensis, a coral native to the Galapagos Archipelago, has successfully established and invaded the Brazilian coast where it modifies native tropical rocky shore and coral reef communities. In order to understand the processes underlying the establishment of T. tagusensis, we tested whether Maxent, a tool for species distribution modeling, based on the native range of T. tagusensis correctly forecasted the invasion range of this species in Brazil. The Maxent algorithm was unable to predict the Brazilian coast as a suitable environment for the establishment of T. tagusensis. A comparison between these models and a principal component analysis (PCA) allowed us to examine the environmental dissimilarity between the two occupied regions (native and invaded) and to assess the species' occupied niche breadth. According to the PCA results, lower levels of chlorophyll‐a and nitrate on the Atlantic coast segregate the Brazilian and Galapagos environments, implying that T. tagusensis may have expanded its realized niche during the invasion process. We tested the possible realized niche expansion in T. tagusensis by assuming that Tubastraea spp. have similar fundamental niches, which was supported by exploring the environmental space of T. coccinea, a tropical‐cosmopolitan congener of T. tagusensis. We believe that the usage of Maxent should be treated with caution, especially when applied to biological invasion (or climate change) scenarios where the target species has a highly localized native (original) distribution, which may represent only a small portion of its fundamental niche, and therefore a violation of a SDM assumption.