Reconstitution of the Anti-Apoptotic Bcl-2 Protein into Lipid Membranes and Biophysical Evidence for Its Detergent-Driven Association with the Pro-Apoptotic Bax Protein

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A₂ (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.     

Formation of electronically excited states during the interaction of p-benzoquinone with hydrogen peroxide

The reaction between p-benzoquinone and H₂O₂ in slightly alkaline solutions yields three major quinoid products that accumulate in the reaction mixture: (a) 2,3-epoxy-p-benzoquinone, (b) 2-hydroxy-p-benzoquinone and (c) p-benzohydroquinone. The reaction is accompanied by photoemission, probably originating from excited triplet 2-hydroxy-p-benzoquinone. These products originate from hydrogen peroxide and hydroxide nucleophilic addition to the C₂?C₃ double bond, as well as secondary redox interactions. The hydroxy substituent and the epoxide ring exert a substantial influence on the electronic distribution in the p-benzoquinone molecule leading to a decrease in the half-wave potential, as compared to the parent p-benzoquinone. The generation of electronically excited states is the result of reactions secondary to the nucleophilic additions involving 2-hydroxy-p-benzosemiquinone, H₂O₂ and hydroxyl radical. The process involves the primary oxidation of 2-hydroxy-p-benzosemiquinone by hydrogen peroxide, followed by oxidation of the semiquinone by hydroxyl radical leading to the formation of the electronically excited quinone. The decay of the excited triplet to the ground state is accompanied by photoemission with maximal intensity at 485–530 nm. Thermodynamic calculations along with an observed increase of photoemission intensity in anaerobiosis point to the triplet (n, π*) multiplicity of the excited state. The efficiency of chemiluminescence could be calculated as 10^?8 photons/2-hydroxy-p-benzoquinone molecule formed. Photoemission arising from the p-benzoquinone/H₂O₂ reaction was inhibited efficiently by addition of GSH to the reaction mixture. This may be due to deactivation of the triplet quinone by a 2-glutathionyl-p-benzohydroquinone adduct, involving thioether α-hydrogen atom-transfer to the triplet ketone.     

Microdetermination of caffeine in blood by gas chromatography—mass spectrometry

A micromethod for the quantitative analysis of caffeine present in small quantities (100 μl) of whole blood is described. It is based on the gas chromatographic—mass spectrometric analysis of chloroform extracts of biological samples. The method is relatively simple, rapid, specific and sensitive, as little as 20 ng of caffeine can be measured.     

Site factors are more important than management for indicator species in semi-natural grasslands in southern Sweden

Plant Ecology - Management of semi-natural grasslands is essential to retain the characteristic diversity of flora and fauna found in these habitats. To maintain, restore or recreate favourable...     

Ecological Consequences of Animal Migration: Prey Partial Migration Affects Predator Ecology and Prey Communities

Ecosystems - Patterns of animal migration and the ecological forces that shape them have been studied for centuries. Yet ecological impacts caused by the migration, such as altered...     

Lifting the veil on the correction of double counting incidents in hybrid life cycle assessment

Life cycle assessment (LCA) and environmentally extended input–output analyses (EEIOA) are two techniques commonly used to assess environmental impacts of an activity/product. Their strengths and weaknesses are complementary, and they are thus regularly combined to obtain hybrid LCAs. A number of approaches in hybrid LCA exist, which leads to different results. One of the differences is the method used to ensure that mixed LCA and EEIOA data do not overlap, which is referred to as correction for double counting. This aspect of hybrid LCA is often ignored in reports of hybrid assessments and no comprehensive study has been carried out on it. This article strives to list, compare, and analyze the different existing methods for the correction of double counting. We first harmonize the definitions of the existing correction methods and express them in a common notation, before introducing a streamlined variant. We then compare their respective assumptions and limitations. We discuss the loss of specific information regarding the studied activity/product and the loss of coherent financial representation caused by some of the correction methods. This analysis clarifies which techniques are most applicable to different tasks, from hybridizing individual LCA processes to integrating complete databases. We finally conclude by giving recommendations for future hybrid analyses.     

Multispectral canopy reflectance improves spatial distribution models of Amazonian understory species

Species distribution models are required for the research and management of biodiversity in the hyperdiverse tropical forests, but reliable and ecologically relevant digital environmental data layers are not always available. We here assess the usefulness of multispectral canopy reflectance (Landsat) relative to climate data in modelling understory plant species distributions in tropical rainforests. We used a large dataset of quantitative fern and lycophyte species inventories across lowland Amazonia as the basis for species distribution modelling (SDM). As predictors, we used CHELSA climatic variables and canopy reflectance values from a recent basin-wide composite of Landsat TM/ETM+ images both separately and in combination. We also investigated how species accumulate over sites when environmental distances were expressed in terms of climatic or surface reflectance variables. When species accumulation curves were constructed such that differences in Landsat reflectance among the selected plots were maximised, species accumulated faster than when climatic differences were maximised or plots were selected in a random order. Sixty-nine species were sufficiently frequent for species distribution modelling. For most of them, adequate SDMs were obtained whether the models were based on CHELSA data only, Landsat data only or both combined. Model performance was not influenced by species’ prevalence or abundance. Adding Landsat-based environmental data layers overall improved the discriminatory capacity of SDMs compared to climate-only models, especially for soil specialist species. Our results show that canopy surface reflectance obtained by multispectral sensors can provide studies of tropical ecology, as exemplified by SDMs, much higher thematic (taxonomic) detail than is generally assumed. Furthermore, multispectral datasets complement the traditionally used climatic layers in analyses requiring information on environmental site conditions. We demonstrate the utility of freely available, global remote sensing data for biogeographical studies that can aid conservation planning and biodiversity management.     

Digital transformation—life cycle assessment of digital services,multifunctional devices and cloud computing

The International Journal of Life Cycle Assessment -