HS1-associated protein X-1 interacts with membrane-bound IgE: impact on receptor-mediated internalization

Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.     

Light stress provokes plastic and elastic modifications in structure and function of photosystem II in camellia leaves

Adaptation of plants to a continuously changing environment may be approached in terms of an optimisation strategy that is dictated by the thermodynamic demand for minimal entropy production. Any change in environmental input is a stressor in the sense that it disturbs optimality. Stress is defined by this disturbance and is therefore relative, with non-stress serving as the reference condition. Because of the thermodynamic demand for optimality, suboptimality forces the system to undergo a state change to a new optimal state. These state changes bring about the adaptation of the system. Any physical or chemical change that occurs during the adaptation processes is denoted as strain. In the present study, the polyphasic rise in chlorophyll (Chl) a fluorescence (OJIP) is used to investigate the behaviour of photosystem II in camellia leaves under different physiological steady states established by adaptation to different light regimes at both room temperature and 10°C. Analysis of the fast fluorescence rise according to the JIP-test allows establishment of structural and functional parameters, providing a quantification of the system's behaviour. The calculated functional parameters are the specific (per reaction center, RC) and phenomenological (per cross section of the sample, CS) energy fluxes for absorption (ABS), trapping (TR) and electron transport (ET). The flux ratios or the yields (the maximum quantum yield of primary photochemistry, φ_p0, the efficiency with which a trapped exciton can move an electron into the electron transport chain, ψ₀, or the probability that an absorbed photon will move an electron into the electron transport chain, ψ_E0 are structural parameters provided by this test, as well as an expression of the concentration of the reaction centers (RC/CS). The photochemical (k_p) and nonphotochemical (k_N) de-excitation rate constants are also calculated. The present study focuses mainly on the response of photosystem II to cyclic environmental changes, denoted as the residual strain. The study investigates the adaptive processes in relation to the regulation of the different functional and structural parameters, as well as reversibility (elasticity) and irreversibility (plasticity). Having screened camellia leaves for several environmental changes, we observed that the various structural parameters undergo modification that differ in both their extent and degree of elasticity, which indicates that different survival strategies are employed in response to stress.     

Exogenous glutamine requirement is confined to late events of T cell activation

Glutamine is required for the proliferation of lymphocytes, but quantitative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM–1 mM) on the in vitro response of human peripheral blood mononuclear cells (PBMC) to a mitogenic anti-CD3 monoclonal antibody (mAb) was investigated. Expression of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequentially analyzed. Proliferation was measured by a ³H-thymidine incorporation assay. mRNA coding for IL-2, IL-2 receptor, IL-4, IL-5, GM-CSF, and IFN-γ was detectable independently from exogenous glutamine provision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD71 as well as the production of IFN-γ were found to require exogenous glutamine supply. In contrast, production of TNF-α could be observed in the absence of glutamine and was increased to a limited extent by exogenous glutamine. The overall lymphocyte response as reflected by entering into the cell cycle and proliferation was directly correlated with the glutamine concentration of the culture medium. Efficient progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM to 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti-CD3 stimulation of a CD4⁺ T cell clone. Altogether, these data underline that a complete cellular immune response depends on an exogenous glutamine supply. Regarding glutamine requirements, they define early, glutamine-independent and late, glutamine-dependent lymphocyte activation stages.     

Inhibitors of adenosine consuming parasites through polymer-assisted solution phase synthesis of lipophilic 5′-amido-5′-deoxyadenosine derivatives

Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed. Thus, a series of 20 adenosine derivatives with a large lipophilic substituent in N⁶-position were prepared in order to evaluate their potential to inhibit the chloroquine resistant Plasmodium falciparum strain K1 in vitro. The rationale for synthesis of these structures was the high probability of interactions with multiple adenosine associated targets and the assumption that a large hydrophobic N⁶-(4-phenoxy)benzyl substitution should allow the molecules to diffuse across parasite membranes. Starting from readily available inosine, the new compounds were prepared as single isomers using a polymer-assisted acylation protocol enabling the straightforward isolation of the target compounds in pure form. Heterocyclic ring systems were synthesized on-bead on Kenner’s safety-catch linker prior to acylation of the scaffold in solution. Most of the highly pure compounds displayed anti-plasmodial activity in the low micromolar or even submicromolar concentration range.     

Patient-tailored cloning of allergens by phage display: Peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10¹¹ cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( -proline)-Sepharose and yielded 30 mg l⁻¹ culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.     

Inferring species interactions from ecological survey data: A mechanistic approach to predict quantitative food webs of seed feeding by carabid beetles

1. Ecological networks are valuable for ecosystem analysis but their use is often limited by a lack of data because many types of ecological interaction, for example, predation, are short‐lived and difficult to observe or detect. While there are different methods for inferring the presence of interactions, they have rarely been used to predict the interaction strengths that are required to construct weighted, or quantitative, ecological networks.
2. Here, we develop a trait‐based approach suitable for inferring weighted networks, that is, with varying interaction strengths. We developed the method for seed‐feeding carabid ground beetles (Coleoptera: Carabidae) although the principles can be applied to other species and types of interaction.
3. Using existing literature data from experimental seed‐feeding trials, we predicted a per‐individual interaction cost index based on carabid and seed size. This was scaled up to the population level to create inferred weighted networks using the abundance of carabids and seeds from empirical samples and energetic intake rates of carabids from the literature. From these weighted networks, we also derived a novel measure of expected predation pressure per seed type per network.
4. This method was applied to existing ecological survey data from 255 arable fields with carabid data from pitfall traps and plant seeds from seed rain traps. Analysis of these inferred networks led to testable hypotheses about how network structure and predation pressure varied among fields.
5. Inferred networks are valuable because (a) they provide null models for the structuring of food webs to test against empirical species interaction data, for example, DNA analysis of carabid gut regurgitates and (b) they allow weighted networks to be constructed whenever we can estimate interactions between species and have ecological census data available. This permits ecological network analysis even at times and in places when interactions were not directly assessed.

     

Transcription under the control of nuclear Arm/beta-catenin

The Wingless/Wnt pathway controls cell fates during animal development and regulates tissue homeostasis as well as stem cell number and differentiation in epithelia. Deregulation of Wnt signaling has been associated with cancer in humans. In the nucleus, the Wingless/Wnt signal is transmitted via the key effector protein Armadillo/beta-catenin. The recent identification and functional analysis of novel Armadillo/beta-catenin interaction partners provide new and exciting insights into the highly complex mechanism of Wingless/Wnt target gene activation.     

Recognition of neurohormones of the NPY family by their receptors

In this review a structural approach developed to answer the question whether hormones from the neuropeptide Y (NPY) family are recognized directly from solution or from the membrane-bound state is described. The chosen strategy is built onto a comparison of a set of peptides with well-known pharmacology and investigates whether similarities of structures of pharmacologically related peptides are higher in solution or in the membrane-bound state. Moreover, we have established the membrane-association mode of these peptides and contributed to our understanding of the structural features of these hormones both when placed in bulk solution and when bound to membranes. As a result we propose a receptor recognition pathway that includes initial association with the membrane and requires the peptides to come off the membrane to diffuse into the binding pocket of the receptor. This review also presents methodology recently developed by us to simulate the structural transition the peptides undergo when diffusing from bulk solution onto the membrane.     

Evolutionary patterns of MHC class II B in owls and their implications for the understanding of avian MHC evolution

Owing to its special mode of evolution and central role in the adaptive immune system, the major histocompatibility complex (MHC) has become the focus of diverse disciplines such as immunology, evolutionary ecology, and molecular evolution. MHC evolution has been studied extensively in diverse vertebrate lineages over the last few decades, and it has been suggested that birds differ from the established mammalian norm. Mammalian MHC genes evolve independently, and duplication history (i.e., orthology) can usually be traced back within lineages. In birds, this has been observed in only 3 pairs of closely related species. Here we report strong evidence for the persistence of orthology of MHC genes throughout an entire avian order. Phylogenetic reconstructions of MHC class II B genes in 14 species of owls trace back orthology over tens of thousands of years in exon 3. Moreover, exon 2 sequences from several species show closer relationships than sequences within species, resembling transspecies evolution typically observed in mammals. Thus, although previous studies suggested that long-term evolutionary dynamics of the avian MHC was characterized by high rates of concerted evolution, resulting in rapid masking of orthology, our results question the generality of this conclusion. The owl MHC thus opens new perspectives for a more comprehensive understanding of avian MHC evolution.     

Novel azabicyclo[3.2.2]nonane derivatives and their activities against Plasmodium falciparum K1 and Trypanosoma brucei rhodesiense

New diaryl substituted 2-azabicyclo[3.2.2]nonane derivatives have been synthesized in order to investigate the influence of the aromatic substitution and of N substitution on the antiprotozoal activities of those compounds. Following a manual method for the Hansch approach, different 4-substituted aryl rings were systematically inserted, and moieties with varying basicity and polarity were attached to the ring nitrogen. All compounds were investigated for their activities against Trypanosoma brucei rhodesiense (STIB 900) and the K(1) strain of Plasmodium falciparum (resistant to chloroquine and pyrimethamine) and for their cytotoxicity using microplate assays. Some of the new compounds are amongst the most active antitrypanosomal agents in this series, and the selectivity index of a single derivative is superior in the 2-azabicyclo-nonane series.