Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells

Echinocytes formed from discocytic erythrocytes by electric field pulses at 0 degrees C return to the discoytic shape upon incubation at 37 degrees C and subsequently turn into stomatocytes. Active and passive components of phospholipid translocation are involved in this shape recovery. Following low-field-strength pulses (5 kV cm-1), shape recovery is fully suppressed by ATPase inhibitors, such as vanadate. When vanadate is only added after stomatocyte formation has been completed, the cells return to the stage of echinocytosis prevailing before recovery. At higher field strength (7 kV cm-1) and in particular after repetitive field pulses, the subsequent incubation at 37 degrees C results in partial shape recovery even in the presence of vanadate. On the basis of the enhanced passive transmembrane mobilities of phospholipid probes observed previously following electroporation, the shape changes in the presence of vanadate are proposed to be due to a passive net movement of phospholipids from the outer to the inner membrane leaflet, as a consequence of the different mobilities of the various membrane phospholipids. Repetitive pulses at higher field strengths lead to a progressively more discocytic stationary shape during subsequent resealing. This phenomenon is explained by the progressively increased transbilayer mobility of the normally almost immobile phospholipid sphingomyelin and a consecutive progressive symmetrization of all membrane phospholipds.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (²H/¹⁵N/¹³C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K_D values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.     

Vegetation controls methane emissions in a coastal brackish fen

Climate change and associated sea level rise will likely affect coastal ecosystems and lead to more frequent inundations. Plants are an important control for methane (CH₄) emissions in peatlands because the metabolism of the living plant can either enhance or attenuate CH₄ emissions and plant litter supplies an easily available carbon source for methanogenesis. Here we compare the contribution of various dominant plant species to methane emissions in a degraded, rewetted coastal brackish fen at the southern Baltic Sea coast in Northeast Germany. We analyse one year of bi-weekly static closed chamber data gathered at measurement spots that were located in different mono-dominant vegetation stands (Bolboschoenus maritimus (L.) Palla, Schoenoplectus tabernaemontani (C.C.Gmel.) Palla, Carex acutiformis Ehrh.). Furthermore, data on water level, water temperature, conductivity (sulphate), and several peat characteristics were recorded. Generally, the annual methane emissions were low with an average across vegetation stands of 14 kg CH₄ ha^?1 a^?1, which we related to high decomposition of peat after drainage and to relatively low water levels in summer. Nevertheless, methane emissions varied between different vegetation types with significantly higher methane fluxes (31.8 ± 5.7 kg CH₄ ha^?1 a^?1) from Bolboschoenus maritimus stands compared to Carex acutiformis and Schoenoplectus tabernaemontani stands (4.3 ± 1.2 and 5.7 ± 2.4 kg CH₄ ha^?1 a^?1, respectively). None of the environmental variables that have been recorded can explain this difference. Thus, vegetation composition seems to be an important driver for methane emissions in coastal brackish fens and may therefore be crucial with regard to recreation measures.     

Morphologically Homogeneous Red Blood Cells Present a Heterogeneous Response to Hormonal Stimulation

Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca²⁺ sensor Fluo-4. Additionally, we developed an approach for analysing the Ca²⁺ responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca²⁺ influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca²⁺ response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca²⁺ revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca²⁺ influx and the associated pro-thrombotic activity.     

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre‐depleted or post‐depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber‐flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.     

Interactions of donor sources and media influence the histo‐morphological quality of full‐thickness skin models

Human artificial skin models are increasingly employed as non‐animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built‐up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo‐morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo‐epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full‐thickness skin models derived from 16 different donors, built‐up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium‐donor‐interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter‐individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium‐donor‐interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.