Interaction of pepsin with aromatic amino acids and their derivatives immobilized to Sepharose

A new LC-MS/MS method for the separation, identification and quantification of residues of 17alpha-estradiol (17alpha-E2) and 17beta-estradiol (17beta-E2) in bovine serum is reported. Deuterium-labelled 17beta-estradiol was used as internal standard. The method was in-house validated in accordance with European Union criteria and adopted in a proficiency study organised by the Community Reference Laboratory (CRL-RIVM, Bilthoven, The Netherlands). The analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a C18 LC column. They were then ionized in a heated nebulizer (HN) interface operating in negative ion mode, where only intact deprotonated molecules, [M-H](-), were generated at m/z 271 and 274 for 17alpha/17beta-E2 and 17beta-E2-d(3), respectively. The decision limits obtained (CCalpha, i.e., critical concentration alpha) were 0.06 ng/mL and 0.03 ng/mL, respectively for 17alpha-E2 and 17beta-E2. Detection capability (CCbeta, i.e., critical concentration beta) values were 0.08 ng/mL and 0.04 ng/mL, respectively, for 17alpha-E2 and 17beta-E2. Precision, accuracy and specificity were satisfactory, recovery ranged from 86.3% to 93.2% and the method resulted sensitive for the required purposes. This method is currently in use for Official Control purposes.     

The species richness–productivity relationship in the herb layer of European deciduous forests

Aim In contrast to non‐forest vegetation, the species richness–productivity (SR‐P) relationship in forests still remains insufficiently explored. Several studies have focused on the diversity of the tree layer, but the species richness of temperate deciduous forests is mainly determined by their species‐rich herb layer. The factors controlling herb‐layer productivity may differ from those affecting tree layers or open herbaceous vegetation, and thus the SR‐P relationship and its underlying processes may differ. However, the few relevant studies have reported controversial results. Here we explore the SR‐P relationship in the forest herb layer across different areas from oceanic to continental Europe, and put the effect of habitat productivity on species richness into context with other key factors, namely soil pH and light availability. Location North‐western Germany, Czech Republic, Slovakia and southern Urals (Russia). Methods We measured herb‐layer species richness and biomass, soil pH and tree‐layer cover in 156 vegetation plots of 100 m² in deciduous forests. We analysed the SR‐P relationship and the relative importance of environmental variables using regression models for particular areas and separate forest types. Results We found a consistent monotonic increase in the herb‐layer species richness with productivity across all study areas and all forest types. Soil pH and light availability also affected species richness, but their relative importance differed among areas. Main conclusions We suggest that the monotonically increasing SR‐P relationship in the forest herb layer results from the fact that herb‐layer productivity is limited by canopy shading; competition within the herb layer is therefore not strong enough to exclude many species. This differs fundamentally from open herbaceous vegetation, which is not subject to such productivity limits and consequently exhibits a unimodal SR‐P relationship. We present a conceptual model that might explain the differences in the SR‐P relationship between the forest herb layer and open herbaceous vegetation.     

Regional differences in soil pH niche among dry grassland plants in Eurasia

Soil pH is a key predictor of plant species occurrence owing to its effect on the availability of nutrients and phytotoxic metals. Although regional differences in realized soil pH niche (‘niche shifts’) have been reported since the 19th century, no study has disentangled how they are influenced by spatial differences in substrate availability, macroclimate, and competitors. We linked plot‐level data on species occurrence and measured soil pH from dry grasslands in eight regions across Eurasia (n = 999 plots), spanning a geographic gradient of 6862 km. We calculated regional shifts in niche optimum (D_opt) and width (D_width) for 73 Species × Region 1 × Region 2 combinations (SRRs; 38 study species) using extended Huisman–Olff–Fresco models. Next, we used commonality analysis to partition the contribution of substrate availability, precipitation, and species traits indicative of competitive ability to variation in regional niche shifts. Shifts in optimum were rare (5% of SRRs with D_opt ≥ 1 pH units) but many species did not show optima within regions. By contrast, shifts in niche width were common (22% of SRRs with D_width ≥1 pH units) and there were pronounced interspecific differences. Whereas none of the three predictors significantly explained shifts in niche optimum, common and unique effects of substrate availability and precipitation accounted for 85% of variation in niche width. Our results suggest that substrate availability and precipitation could be the driving factors behind species regional shifts in niche width. Studies that address additional factors, such as other edaphic niches, and their variability at the regional and micro‐scale will improve our understanding of the mechanisms underlying species distributions.     

The invasion history,distribution and colour pattern forms of the harlequin ladybird beetle Harmonia axyridis (Pall.) (Coleoptera,Coccinellidae) in Slovakia,Central Europe

The harlequin ladybird beetle Harmonia axyridis (Coleoptera, Coccinellidae) has invaded and established in Slovakia. Following unintentional introduction in 2008, the spread of the alien coccinellid was very fast. By the end of 2009, it was recorded across the whole country, and by the end of 2012 it was widely distributed and common in various habitats, particularly gardens, orchards and urban areas, where it was most frequent on trees. The rate of eastward spread was approximately 200 km year^-1, similar to the overall rate of spread in Europe. Between 2008 and 2012, the coccinellid was recorded in a total of 153 localities, in altitudes ranging from 98 to 1,250 m. Most records of this species were made in lowlands, hilly areas and valleys separating mountain ridges. However, it was only rarely documented in areas above 700 m a.s.l. The non-melanic colour form (f. succinea) was dominant along a longitudinal transect including eight urban areas across Slovakia, with the frequency of melanic forms (f. spectabilis and f. conspicua together) between 6.3 and 19.2% and a median equal to 10.5%. The invasion history and distribution of H. axyridis in Slovakia are discussed with regard to the time sequence of records, rate of spread, altitudinal distribution, anthropogenic dispersal, effective recording, proportion of melanic forms and other relevant aspects associated with the spread of this successful invader.     

How climate change affects the occurrence of a second generation in the univoltine Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

1. Understanding the conditions that allow for the occurrence of an additional generation in populations that are usually univoltine is important under the present climate warming. In temperate areas, a second generation is enabled through the emergence of a time window that opens when first-generation individuals are ready to reproduce and closes when second-generation individuals cannot complete development before the onset of winter. 2. The conditions that limit the width of this window were studied in Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae), a ground-inhabiting heteropteran overwintering in facultative adult diapause, whose populations in Central Europe have typically been univoltine until the 1980s. 3. The frequency of females of the first generation that started to lay eggs decreased from 70% in June to zero in early August, but oviposition of these females continued until the end of August. Using thermal constants for egg–adult development and temperature data, this study found that the development of most second-generation individuals could only be completed before the start of winter if hastened through behavioural thermoregulation. 4. Consequences of temperature increase on the width of the thermal window were calculated. Increasing temperature causes the time window to open earlier and close later by accelerating maturation of first-generation females and improving conditions for maturing of the second-generation individuals in late summer and autumn. 5. Climate warming will create conditions that facilitate the occurrence of a second generation in a year in typically univoltine populations of this species.     

As2O3 oxidation by vitamin C: cell culture studies

The ability of As₂O₃ to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As₂O₃ (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 μmol l⁻¹) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.     

Arsenic trioxide versus tetraarsenic oxide in biomedical research: misunderstandings and misinterpretations

This work presents an analytical chemist’s view on the sometimes unconscious use of arsenic trioxide in (bio)medical research. Arsenic trioxide is a frequently used chemical in cancer treatment research and its action to various malignant cells has been extensively studied and published. Unfortunately some research articles show trivial errors with regards to background knowledge of the chemical, handling the chemical, experimental design and interpretation of results like e.g. in a range of articles comparing advantages of tetraarsenic oxide over arsenic trioxide (dimeric/monomeric) although the dissolution of both yields the same active compound (HAsO₂). To fully understand the implications of these errors we will highlight some of them with the intent to harmonize future work in this field.