Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H⁺] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562?nm was linear in the range pH?=?4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30?μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N?=?28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.     

Early post-fire regeneration of a Pinus halepensis forest on Mount Párnis,Greece

Abstract. The post-fire regeneration of a 45-yr-old Pinus halepensis (Aleppo pine) forest, burned in July 1989, has been studied on Mount Párnis, Attiki, Greece. Four experimental plots at various slopes and exposures were established at altitudes of 400 - 450 m, and monitored for 3 yr at 3-month intervals. Early regeneration took place abundantly, through both resprouting and seed germination of mostly hard-seeded herbs and shrubs; the floristic richness was high with 80 taxa. Pine seedling emergence took place during the winter of the first post-fire year. The mean pine seedling density by the end of the recruitment period (March 1990) was 5–6 seedlings/m². This density decreased slightly during late spring and considerably during summer. During the second post-fire year only a relatively slight decline was observed; thereafter the density was stabilized to 1 - 2 seedlings/m². Mortality follows a negative exponential curve that levels off at ca. 20 %. Height distributions throughout the three post-fire years were all positively skewed as a result of the presence of few very tall saplings. A considerable fraction (20 %) of very short (5–15 cm) saplings were still alive 39 months after the fire; these may constitute the sapling bank. Based on the analysis of height distribution curves, it is concluded that the taller seedlings survived significantly better than the shorter ones.     

Spatial averaging and disturbance lead to high productivity in aquatic metacommunities

Dispersal in heterogeneous ecosystems, such as coastal metacommunities, is a major driver of diversity and productivity. According to theory, both species richness and spatial averaging shape a unimodal relationship of productivity with dispersal. We experimentally tested the hypothesis that disturbances acting on local patches would buffer the loss of productivity at high dispersal by preventing synchronized species oscillations. To simulate these disturbances, our experimental assemblages involved species that self‐organized in isolation under three inflow pulsing frequencies, where hydraulic displacement and nutrient loading affected assemblage diversity and composition. At steady‐state, the emerging isolated assemblages were connected at three levels of dispersal creating three metacommunities of different connectivity. Consistent with theory, as dispersal increased, species richness in the metacommunity declined; productivity however remained high. This occurred because the most productive species in our study (which dominated the isolated patch of intermediate inflow pulsing frequency) dominated all three patches (low, intermediate and high inflow pulsing frequencies) after dispersal commenced in our metacommunities. This experimental result provides empirical support for the mechanism of spatial averaging. Furthermore, disturbances, in the form of localized pulsed inflows, prevented population oscillation synchrony caused by homogenization. Overall, our observations suggest that localized environmental fluctuations and the identity of species seem to be more influential than dispersal in shaping the diversity and composition of phytoplankton assemblages and stabilizing productivity.     

Herbivore species identity rather than diversity of the non‐host community determines foraging behaviour of the parasitoid wasp Cotesia glomerata

Extensive research has been conducted to reveal how species diversity affects ecosystem functions and services. Yet, consequences of diversity loss for ecosystems as a whole as well as for single community members are still difficult to predict. Arthropod communities typically are species‐rich, and their species interactions, such as those between herbivores and their predators or parasitoids, may be particularly sensitive to changes in community composition. Parasitoids forage for herbivorous hosts by using herbivore‐induced plant volatiles (indirect cues) and cues produced by their host (direct cues). However, in addition to hosts, non‐suitable herbivores are present in a parasitoid's environment which may complicate the foraging process for the parasitoid. Therefore, ecosystem changes in the diversity of herbivores may affect the foraging efficiency of parasitoids. The effect of herbivore diversity may be mediated by either species numbers per se, by specific species traits, or by both. To investigate how diversity and identity of non‐host herbivores influence the behaviour of parasitoids, we created environments with different levels of non‐host diversity. On individual plants in these environments, we complemented host herbivores with 1–4 non‐host herbivore species. We subsequently studied the behaviour of the gregarious endoparasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) while foraging for its gregarious host Pieris brassicae L. (Lepidoptera: Pieridae). Neither non‐host species diversity nor non‐host identity influenced the preference of the parasitoid for herbivore‐infested plants. However, after landing on the plant, non‐host species identity did affect parasitoid behaviour, whereas non‐host diversity did not. One of the non‐host species, Trichoplusia ni Hübner (Lepidoptera: Noctuidae), reduced the time the parasitoid spent on the plant as well as the number of hosts it parasitized. We conclude that non‐host herbivore species identity has a larger influence on C. glomerata foraging behaviour than non‐host species diversity. Our study shows the importance of species identity over species diversity in a multitrophic interaction of plants, herbivores, and parasitoids.     

The role of phospholamban in the regulation of calcium transport by cardiac sarcoplasmic reticulum

The calcium transport mechanism of cardiac sarcoplasmic reticulum (SR) is regulated by a phosphoregulatory mechanism involving the phosphorylation-dephosphorylation of an integral membrane component, termed phospholamban. Phospholamban, a 27,000 Da proteolipid, contains phosphorylation sites for three independent protein kinases: 1) cAMP-dependent, 2) Ca²⁺-calmodulin-dependent, and 3) Ca²⁺-phospholipid-dependent. Phosphorylation of phospholamban by any one of these kinases is associated with stimulation of the calcium transport rates in isolated SR vesicles. Dephosphorylation of phosphorylated phospholamban results in the reversal of the stimulatory effects produced by the protein kinases. Studies conducted on perfused hearts have shown that during exposure to beta-adrenergic agents, a good correlation exists between the in situ phosphorylation of phospholamban and the relaxation of the left ventricle. Phosphorylation of phospholamban in situ is also associated with stimulation of calcium transport rates by cardiac SR, similar to in vitro findings. Removal of beta-adrenergic agents results in the reversal of the inotropic response and this is associated with dephosphorylation of phospholamban. These findings indicate that a phospho-regulatory mechanism involving phospholamban may provide at least one of the controls for regulation of the contractile properties of the myocardium.     

Comparison of bioactivity between GSM 900 MHz and DCS 1800 MHz mobile telephony radiation

An increasing number of studies find that pulsed Radio Frequency (RF), electromagnetic radiation of both systems of digital mobile telephony, established and commonly used in Europe during the last years, GSM 900 MHz (Global System for Mobile telecommunications) and DCS 1800 MHz (Digital Cellular System), exert intense biological action on different organisms and cells (Hardell et al., 2006; Hyland, 2000; Kundi, 2004; Panagopoulos et al., 2004, 2007). The two types of cellular telephony radiation use different carrier frequencies and give different frequency spectra, but they usually also differ in intensity, as GSM 900 MHz antennas operate at about double the power output than the corresponding DCS 1800 MHz ones. In our present experiments, we used a model biological system, the reproductive capacity of Drosophila melanogaster, to compare the biological activity between the two systems of cellular mobile telephony radiation. Both types of radiation were found to decrease significantly and non thermally the insect's reproductive capacity, but GSM 900 MHz seems to be even more bioactive than DCS 1800 MHz. The difference seems to be dependent mostly on field intensity and less on carrier frequency.     

Ankyrin-B syndrome: enhanced cardiac function balanced by risk of cardiac death and premature senescence

Here we report the unexpected finding that specific human ANK2 variants represent a new example of balanced human variants. The prevalence of certain ANK2 (encodes ankyrin-B) variants range from 2 percent of European individuals to 8 percent in individuals from West Africa. Ankyrin-B variants associated with severe human arrhythmia phenotypes (eg E1425G, V1516D, R1788W) were rare in the general population. Variants associated with less severe clinical and in vitro phenotypes were unexpectedly common. Studies with the ankyrin-B(+/-) mouse reveal both benefits of enhanced cardiac contractility, as well as costs in earlier senescence and reduced lifespan. Together these findings suggest a constellation of traits that we term "ankyrin-B syndrome", which may contribute to both aging-related disorders and enhanced cardiac function.     

C-Glucosylated malonitrile as a key intermediate towards carbohydrate-based glycogen phosphorylase inhibitors

Glycogen utilization involves glycogen phosphorylase, an enzyme which appears to be a potential target for the regulation of glycaemia, as the liver isoform is a major player for hepatic glucose output. A single C-glucosylated malonitrile allowed for the synthesis of three glucose-based derivatives namely bis-oxadiazoles, bis-amides and a C-glucosylated tetrahydropyrimidin-2-one. When evaluated as glycogen phosphorylase inhibitors, two of the synthesized compounds displayed inhibition in the sub-millimolar range. In silico studies revealed that only one out of the bis-amides obtained and the C-glucosylated tetrahydropyrimidin-2-one may bind at the catalytic site.