Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Promotion of liver regeneration by natural killer cells in a murine model is dependent on extracellular adenosine triphosphate phosphohydrolysis

Nucleotides, such as adenosine triphosphate (ATP), are released by cellular injury, bind to purinergic receptors expressed on hepatic parenchymal and nonparenchymal cells, and modulate cellular crosstalk. Liver resection and resulting cellular stress initiate such purinergic signaling responses between hepatocytes and innate immune cells, which regulate and ultimately drive liver regeneration. We studied a murine model of partial hepatectomy using immunodeficient mice to determine the effects of natural killer (NK) cell-mediated purinergic signaling on liver regeneration. We noted first that liver NK cells undergo phenotypic changes post-partial hepatectomy (PH) in vivo, including increased cytotoxicity and more immature phenotype manifested by alterations in the expression of CD107a, CD27, CD11b, and CD16. Hepatocellular proliferation is significantly decreased in Rag2/common gamma-null mice (lacking T, B, and NK cells) when compared to wildtype and Rag1-null mice (lacking T and B cells but retaining NK cells). Extracellular ATP levels are elevated post-PH and NK cell cytotoxicity is substantively increased in vivo in response to hydrolysis of extracellular ATP levels by apyrase (soluble NTPDase). Moreover, liver regeneration is significantly increased by the scavenging of extracellular ATP in wildtype mice and in Rag2/common gamma-null mice after adoptive transfer of NK cells. Blockade of NKG2D-dependent interactions significantly decreased hepatocellular proliferation. In vitro, NK cell cytotoxicity is inhibited by extracellular ATP in a manner dependent on P2Y1, P2Y2, and P2X3 receptor activation. Conclusion: We propose that hepatic NK cells are activated and cytotoxic post-PH and support hepatocellular proliferation. NK cell cytotoxicity is, however, attenuated by hepatic release of extracellular ATP by way of the activation of specific P2 receptors. Clearance of extracellular ATP elevates NK cell cytotoxicity and boosts liver regeneration.

Annexins sense changes in intracellular pH during hypoxia

The pH(i) (intracellular pH) is an important physiological parameter which is altered during hypoxia and ischaemia, pathological conditions accompanied by a dramatic decrease in pH(i). Sensors of pH(i) include ion transport systems which control intracellular Ca2+ gradients and link changes in pH(i) to functions as diverse as proliferation and apoptosis. The annexins are a protein family characterized by Ca2+-dependent interactions with cellular membranes. Additionally, in vitro evidence points to the existence of pH-dependent, Ca(2+)-independent membrane association of several annexins. We show that hypoxia promotes the interaction of the recombinant annexin A2-S100A10 (p11) and annexin A6 with the plasma membrane. We have investigated in vivo the influence of the pH(i) on the membrane association of human annexins A1, A2, A4, A5 and A6 tagged with fluorescent proteins, and characterized this interaction for endogenous annexins present in smooth muscle and HEK (human embryonic kidney)-293 cells biochemically and by immunofluorescence microscopy. Our results show that annexin A6 and the heterotetramer A2-S100A10 (but not annexins A1, A4 and A5) interact independently of Ca2+ with the plasma membrane at pH 6.2 and 6.6. The dimerization of annexin A2 within the annexin A2-S100A10 complex is essential for the pH-dependent membrane interaction at this pH range. The pH-induced membrane binding of annexins A6 and A2-S100A10 might have consequences for their functions as membrane organizers and channel modulators.

Enhanced non-perturbative effects through the collinear anomaly

We show that nonperturbative effects are logarithmically enhanced for transverse-momentum-dependent observables such as qT spectra of electroweak bosons in hadronic collisions and jet broadening at e+e− colliders. This enhancement arises from the collinear anomaly, a mechanism characteristic for transverse observables, which induces logarithmic dependence on the hard scale in the product of the soft and collinear matrix elements. Our analysis is based on an operator product expansion and provides, for the first time, a systematic, model-independent way to study nonperturbative effects for this class of observables. For the case of jet broadening, we relate the leading correction to the nonperturbative shift of the thrust distribution.

Probing interactions of NMD factors in a distance‐dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-­‐protein interaction in vivo. For large multi-­‐factor complexes like those assembling on mRNA, co-­‐immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-­‐protein interaction network for key factors in the nonsense-­‐mediated mRNA decay (NMD) pathway in a distant-­‐dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-­‐protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance

Abstract Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

Interactions between dorsolateral and ventromedial prefrontal cortex underlie context-dependent stimulus valuation in goal-directed choice.

External circumstances and internal bodily states often change and require organisms to flexibly adapt valuation processes to select the optimal action in a given context. Here, we investigate the neurobiology of context-dependent valuation in 22 human subjects using functional magnetic resonance imaging. Subjects made binary choices between visual stimuli with three attributes (shape, color, and pattern) that were associated with monetary values. Context changes required subjects to deviate from the default shape valuation and to integrate a second attribute in order to comply with the goal to maximize rewards. Critically, this binary choice task did not involve any conflict between opposing monetary, temporal, or social preferences. We tested the hypothesis that interactions between regions of dorsolateral and ventromedial prefrontal cortex (dlPFC; vmPFC) implicated in self-control choices would also underlie the more general function of context-dependent valuation. Consistent with this idea, we found that the degree to which stimulus attributes were reflected in vmPFC activity varied as a function of context. In addition, activity in dlPFC increased when context changes required a reweighting of stimulus attribute values. Moreover, the strength of the functional connectivity between dlPFC and vmPFC was associated with the degree of context-specific attribute valuation in vmPFC at the time of choice. Our findings suggest that functional interactions between dlPFC and vmPFC are a key aspect of context-dependent valuation and that the role of this network during choices that require self-control to adjudicate between competing outcome preferences is a specific application of this more general neural mechanism.

Thiacloprid–Nosema ceranae interactions in honey bees: Host survivorship but not parasite reproduction is dependent on pesticide dose

Interactions between stressors contribute to the recently reported increase in losses of honey bee colonies. Here we demonstrated that a synergistic effect on mortality by the low toxic, commonly used neonicotinoid thiacloprid and the nearly ubiquitous gut parasite Nosemaceranae is dependent on the pesticide dose. Furthermore, thiacloprid had a negative influence on N.ceranae reproduction. Our results highlight that interactions among honey bee health stressors can be dynamic and should be studied across a broader range of combinations.

IL-33 promotes an innate immune pathway of intestinal tissue protection dependent on amphiregulin-EGFR interactions

The barrier surfaces of the skin, lung, and intestine are constantly exposed to environmental stimuli that can result in inflammation and tissue damage. Interleukin (IL)-33-dependent group 2 innate lymphoid cells (ILC2s) are enriched at barrier surfaces and have been implicated in promoting inflammation; however, the mechanisms underlying the tissue-protective roles of IL-33 or ILC2s at surfaces such as the intestine remain poorly defined. Here we demonstrate that, following activation with IL-33, expression of the growth factor amphiregulin (AREG) is a dominant functional signature of gut-associated ILC2s. In the context of a murine model of intestinal damage and inflammation, the frequency and number of AREG-expressing ILC2s increases following intestinal injury and genetic disruption of the endogenous AREG-epidermal growth factor receptor (EGFR) pathway exacerbated disease. Administration of exogenous AREG limited intestinal inflammation and decreased disease severity in both lymphocyte-sufficient and lymphocyte-deficient mice, revealing a previously unrecognized innate immune mechanism of intestinal tissue protection. Furthermore, treatment with IL-33 or transfer of ILC2s ameliorated intestinal disease severity in an AREG-dependent manner. Collectively, these data reveal a critical feedback loop in which cytokine cues from damaged epithelia activate innate immune cells to express growth factors essential for ILC-dependent restoration of epithelial barrier function and maintenance of tissue homeostasis.

Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID)

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC (1)H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS. (C) 2011 Elsevier B.V. All rights reserved.

Measurement of charged-particle event shape variables in inclusive √(s)=7 TeV proton-proton interactions with the ATLAS detector

The measurement of charged-particle event shape variables is presented in inclusive inelastic pp collisions at a center-of-mass energy of 7 TeV using the ATLAS detector at the LHC. The observables studied are the transverse thrust, thrust minor, and transverse sphericity, each defined using the final-state charged particles' momentum components perpendicular to the beam direction. Events with at least six charged particles are selected by a minimum-bias trigger. In addition to the differential distributions, the evolution of each event shape variable as a function of the leading charged-particle transverse momentum, charged-particle multiplicity, and summed transverse momentum is presented. Predictions from several Monte Carlo models show significant deviations from data.