Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR.

The Antennapedia homeodomain protein of Drosophila has the ability to penetrate biological membranes and the third helix of this protein, residues 43-58, known as penetratin (RQIKIWFQNRRMKWKK-amide) has the same translocating properties as the entire protein. The variant, RQI KIFFQNRRMKFKK-amide, here called penetratin (W48F,W56F) does not have the same ability. We have determined a solution structure of penetratin and investigated the position of both peptides in negatively charged bicelles. A helical structure is seen for residues Lys46 through Met54. The secondary structure of the variant penetratin(W48F,W56F) in bicelles appears to be very similar. Paramagnetic spin-label studies and analysis of NOEs between penetratin and the phospholipids show that penetratin is located within the bicelle surface. Penetratin (W48F,W56F) is also located inside the phospholipid bicelle, however, with its N-terminus more deeply inserted than that of wild-type penetratin. The subtle differences in the way the two peptides interact with a membrane in an equilibrium situation could be important for their translocating ability. As a comparison we have also investigated the secondary structure of penetratin(W48F,W56F) in SDS micelles and the results show that the structure is very similar in SDS and bicelles. In contrast, penetratin(W48F,W56F) and penetratin appear to be located differently in SDS micelles. This clearly shows the importance of using realistic membrane mimetics for investigating peptide-membrane interactions.     

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.     

High-throughput system for determining dissolution kinetics of inclusion bodies

Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01–0.03 s^–1 for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more.     

Vaccinia Virus-Mediated Inhibition of Type I Interferon Responses Is a Multifactorial Process Involving the Soluble Type I Interferon Receptor B18 and Intracellular Components

Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not. These results implied that VACV expressed an IFN inhibitor(s) that was functionally deleted in MVA. To further characterize the IFN inhibitor(s), infection experiments were carried out with CVA strains isolated after 152 (CVA152) and 386 CEF passages (CVA386). Interestingly, neither CVA152 nor CVA386 induced IFN-α, whereas the latter variant did induce IFN-β. This pattern suggested a consecutive loss of inhibitors during MVA attenuation. Similar to supernatants of VACV- and CVA152-infected DC cultures, recombinantly expressed soluble IFN decoy receptor B18, which is encoded in the VACV genome, inhibited MVA-induced IFN-α but not IFN-β. In the same direction, a B18R-deficient VACV variant triggered only IFN-α, confirming B18 as the soluble IFN-α inhibitor. Interestingly, VACV infection inhibited IFN responses induced by a multitude of different stimuli, including oligodeoxynucleotides containing CpG motifs, poly(I:C), and vesicular stomatitis virus. Collectively, the data presented show that VACV-mediated IFN inhibition is a multistep process involving secreted factors such as B18 plus intracellular components that cooperate to efficiently shut off systemic IFN-α and IFN-β responses.     

Solid-State NMR Evidence for Inequivalent GvpA Subunits in Gas Vesicles

Gas vesicles are organelles that provide buoyancy to the aquatic microorganisms that harbor them. The gas vesicle shell consists almost exclusively of the hydrophobic 70-residue gas vesicle protein A, arranged in an ordered array. Solid-state NMR spectra of intact collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae show duplication of certain gas vesicle protein A resonances, indicating that specific sites experience at least two different local environments. Interpretation of these results in terms of an asymmetric dimer repeat unit can reconcile otherwise conflicting features of the primary, secondary, tertiary, and quaternary structures of the gas vesicle protein. In particular, the asymmetric dimer can explain how the hydrogen bonds in the β-sheet portion of the molecule can be oriented optimally for strength while promoting stabilizing aromatic and electrostatic side-chain interactions among highly conserved residues and creating a large hydrophobic surface suitable for preventing water condensation inside the vesicle.     

Role of bradykinin in gastric vasodilation caused by hypertonic saline and acid back diffusion

Protective vasodilation in response to tissue injury and acid back diffusion is associated with release of bradykinin in the rat stomach. We hypothesized that bradykinin might be involved in mechanisms behind such vasodilation via influence on mast cells and sensory neurons. Acid back diffusion after mucosal barrier disruption with hypertonic saline evoked degranulation of mast cells in the rat stomach wall. Acid back diffusion was also associated with increased luminal release of histamine and gastric blood flow in normal rats, but not in mast cell-deficient rats. Bradykinin (BK(2)) receptor blockade inhibited degranulation of submucosal mast cells in the stomach and attenuated gastric vasodilation both in response to acid back diffusion and after stimulation of sensory neurons with capsaicin. Gastric vasodilation caused by mucosal injury with hypertonic saline alone was associated with degranulation of mucosal mast cells. These events were unaffected by inhibition of prostaglandin synthesis, whereas bradykinin (BK(2)) receptor blockade was associated with abolished vasodilation and inhibition of mucosal mast cell degranulation. We conclude that bradykinin is involved in gastric vasodilation caused by hypertonic injury alone via influence on mast cells, and by acid back diffusion via influence on both sensory neurons and mast cells.     

Effect of phytosterols and phytostanols on the solubilization of cholesterol by dietary mixed micelles: an in vitro study

The effect of a plant sterol, beta-sitosterol (SI), and a plant stanol, sitostanol (SS), on the solubilization of cholesterol (CH) by model dietary mixed micelles was examined under in vitro conditions with the use of gas chromatography, isothermal titration calorimetry, NMR spectroscopy and cryogenic transmission electron microscopy techniques. Free SI and SS were shown to reduce the concentration of CH in dietary mixed micelles via a dynamic competition mechanism. CH, SI and SS affect the microstructure of lipid vesicles and influence the process of amphiphilic self-assembly of nutrients in the gut with the formation of dietary mixed micelles in a similar manner. Therefore, substitution of CH by phytosterols and phytostanols in the diet does not lead to the notable changes in the mechanism of dietary mixed micelle formation and does not affect the process of the intestinal transport of nutrients and drugs via the micellar diffusion mechanism. Our experimental findings demonstrate that the introduction of plant sterols and plant stanols into the diet is clearly beneficial for the reduction of the intestinal uptake of cholesterol. Due to the limited capacity of dietary mixed micelles to embody hydrophobic sterol/stanol molecules, the micellar concentration of cholesterol is reduced and hence, its transport towards the intestinal brush border membrane decreases.     

Climate warming and precipitation redistribution modify tree–grass interactions and tree species establishment in a warm‐temperate savanna

Savanna tree–grass interactions may be particularly sensitive to climate change. Establishment of two tree canopy dominants, post oak (Quercus stellata) and eastern redcedar (Juniperus virginiana), grown with the dominant C₄ perennial grass (Schizachyrium scoparium) in southern oak savanna of the United States were evaluated under four climatic scenarios for 6 years. Tree–grass interactions were examined with and without warming (+1.5 °C) in combination with a long‐term mean rainfall treatment and a modified rainfall regime that redistributed 40% of summer rainfall to spring and fall, intensifying summer drought. The aim was to determine: (1) the relative growth response of these species, (2) potential shifts in the balance of tree–grass interactions, and (3) the trajectory of juniper encroachment into savannas, under these anticipated climatic conditions. Precipitation redistribution reduced relative growth rate (RGR) of trees grown with grass. Warming increased growth of J. virginiana and strongly reduced Q. stellata survival. Tiller numbers of S. scoparium plants were unaffected by warming, but the number of reproductive tillers was increasingly suppressed by intensified drought each year. Growth rates of J. virginiana and Q. stellata were suppressed by grass presence early, but in subsequent years were higher when grown with grass. Quercus stellata had overall reduced RGR, but enhanced survival when grown with grass, while survival of J. virginiana remained near 100% in all treatments. Once trees surpassed a threshold height of 1.1 m, both tiller number and survival of S. scoparium plants were drastically reduced by the presence of J. virginiana, but not Q. stellata. Juniperus virginiana was the only savanna dominant in which neither survival nor final aboveground mass were adversely affected by the climate scenario of warming and intensified summer drought. These responses indicate that climate warming and altered precipitation patterns will further accelerate juniper encroachment and woody thickening in a warm‐temperate oak savanna.     

The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.