Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.     

Increased Cation Conductance in Human Erythrocytes Artificially Aged by Glycation

Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which may result from activation of Ca²⁺ permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca²⁺]_i from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca²⁺ channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30–48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37°C) significantly increased glycation of membrane proteins, hemoglobin (HbA_1c), TRPC3/6/7, and L-type Ca²⁺ channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca²⁺]_i, and phosphatidylserine exposure, and led to significant cell shrinkage. Ca²⁺ removal and addition of Ca²⁺ chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca²⁺ entry from extracellular space.     

Development, ultrastructure, natural occurrence, and molecular characterization of Liebermania patagonica n. g., n. sp., a microsporidian parasite ot the grasshopper Tristira magellanica (Orthoptera: Tristiridae)

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.     

Combined modulation of the mitochondrial ATP-dependent potassium channel and the permeability transition pore causes prolongation of the biphasic calcium dynamics

The permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel of mitochondria are known to play key roles in mitochondrially mediated apoptosis. We investigated how modulation of the permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel, either as single elements or in combination, affects the proapoptotic intracellular calcium ([Ca(2+)](i)) transients and the mitochondrial membrane potential (psi(m)). For this purpose a model was established exploring the [Ca(2+)](i) transients in N2A cells using continuous application of ATP that causes a biphasic [Ca(2+)](i) response. This response was sensitive to endoplasmatic reticulum (ER) Ca(2+) depletion and a smooth ER Ca(2+)-ATPase (SERCA) antagonist. PTP inhibition by cyclosporine A (CsA) or its non-immunosuppressive derivative NIM811 caused an amplification of the secondary [Ca(2+)](i) peak and induced a hyperpolarization of psi(m). Both the putative mtK-ATP channel inhibitor 5-hydroxydecanoate (5-HD) and the opener diazoxide ameliorated the ATP-induced secondary [Ca(2+)](i) peak. The effect of diazoxide was accompanied by a depolarization of psi(m) whereas 5-HD had no effect on psi(m). When diazoxide and CsA or NIM811 were applied together the secondary [Ca(2+)](i) rise did not return to baseline and a not significant hyperpolarization of psi(m) was observed. So, simultaneous inhibition of PTP and activation of the mtK-ATP channel prevents the increased slope of the secondary [Ca(2+)](i) peak induced by CsA (or NIM811) and also the depolarization after diazoxide application. Hence, we propose that modulation of one of these channels leads to functional changes of the other channel by means of Delta[Ca(2+)](i) and Deltapsi(m).     

Modulation of the multilamellar membrane organization and of the chiral macrodomains in the diatom Phaeodactylum tricornutum revealed by small-angle neutron scattering and circular dichroism spectroscopy

Diatoms possess effective photoprotection mechanisms, which may involve reorganizations in the photosynthetic machinery. We have shown earlier, by using circular dichroism (CD) spectroscopy, that in Phaeodactylum tricornutum the pigment-protein complexes are arranged into chiral macrodomains, which have been proposed to be associated with the multilamellar organization of the thylakoid membranes and shown to be capable of undergoing light-induced reversible reorganizations (Szabó et al. Photosynth Res 95:237, 2008). Recently, by using small-angle neutron scattering (SANS) on the same algal cells we have determined the repeat distances and revealed reversible light-induced reorganizations in the lamellar order of thylakoids (Nagy et al. Biochem J 436:225, 2011). In this study, we show that in moderately heat-treated samples, the weakening of the lamellar order is accompanied by the diminishment of the psi-type CD signal associated with the long-range chiral order of the chromophores (psi, polymer or salt-induced). Further, we show that the light-induced reversible increase in the psi-type CD is associated with swelling in the membrane system, with magnitudes larger in high light than in low light. In contrast, shrinkage of the membrane system, induced by sorbitol, brings about a decrease in the psi-type CD signal; this shrinkage also diminishes the non-photochemical quenching capability of the cells. These data shed light on the origin of the psi-type CD signal, and confirm that both CD spectroscopy and SANS provide valuable information on the macro-organization of the thylakoid membranes and their dynamic properties; these parameters are evidently of interest with regard to the photoprotection in whole algal cells.     

Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation

Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.     

Phylogenetic relationships of Heterovesicula cowani, a microsporidian pathogen of Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae), based on SSU rDNA-sequence analyses

The microsporidium Heterovesicula cowani, discovered in 1985, was initially identified as Vairimorpha sp. because it produces two types of spores: Nosema-like diplokaryotic spores and Thelohania-like mononuclear meiospores. However, light and electron microscopy studies revealed characters that did not fit any known microsporidian genera, and a new monotypic genus Heterovesicula was erected. The goal of this study was to test the validity of the genus Heterovesicula by molecular characterization of H. cowani and to assess its phylogenetic relationships to other microsporidia from insects. DNA from spores stored at −32 °C since 1992 was isolated and PCR-amplified with V1-1492 primers to obtain a partial small subunit ribosomal RNA gene sequence of 1165 bp, which was submitted to GenBank (Accession No. EU275200). Neighbor joining, maximum parsimony and maximum likelihood analyses performed against 18 microsporidia sequences, placed H. cowani as a sister taxon to the Nosema–Vairimorpha clade. The consensus of these analyses suggests that the Heterovesicula–Nosema–Vairimorpha group forms a dichotomy with the Encephalitozoon spp. branch. Other microsporidia parasitizing Orthoptera fell into two unrelated (or distantly related) lineages of terrestrial microsporidia: the Liebermannia spp. branch forms a dichotomy with Orthosomella operophterae within the Endoreticulatus–Orthosomella–Liebermannia group; and the Paranosema spp. branch clusters together with the Tubulinosema–Systenostrema lineage. The minimum pairwise distance in Kimura-2-Parameter analysis among 18 analyzed sequences was 0.37, which supports well the generic status for Heterovesicula. The obtained phylogenetic trees suggest that H. cowani is related to the Vairimorpha necatrix group, but not to other insect microsporidia producing octospores.