Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.     

Steroid radiommuoassay: contribution of standards to blank values

The sensitivity of the radioimmunoassay of steroids is considerably reduced by high blank values which may be derived in part from co-chromatographed standards. Blank levels approach the detection limit of the radioimmunoassay of aldosterone, testosterone and androstenedione when 10,000 dpm (30–35 pg) labeled steroids are used as reference standard. When 20 μg aldosterone, testosterone, or androstenedione is used as standard, blank levels of up to 12,800 pg were measured in the radioimmunoassay. Application of the standards on a separate strip does not improve the results. From our experiments it seems clear that contamination is transported by the solvent.     

Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein

Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET(+)) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET(+) and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET(+)-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET(+) produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors.     

Comparison of the Munich Chronotype Questionnaire with the Horne-Ostberg's Morningness-Eveningness Score

We report on results from an Internet survey of sleeping habits in a Dutch population using the Munich Chronotype Questionnaire (MCTQ), supplemented with the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ). The MCTQ was completed by 5,055 responders, of which 2,481 also completed the MEQ. MEQ score correlated well with the MCTQ assessment of time of mid-sleep on free days (MSF; r = - 0.73) and on workdays (MSW; r = - 0.61). MEQ was more strongly correlated with MSF (50% of sleep time) than with sleep onset (0%), rise time (100%), or with any other percentile (10 to 40, 60% to 90%) of sleep on free days. The study shows that chronotype (based on MSF as measured by the MCTQ) strongly correlates with morningness-eveningness (as measured by the MEQ). However, the MCTQ collects additional detailed information on sleep-wake behavior under natural conditions.     

Calcium,a pivotal player in photodynamic therapy?

Photodynamic therapy combines three non-toxic components: light, oxygen and a photosensitizer to generate singlet oxygen and/or other ROS molecules in order to target destruction of cancer cells. The damage induced in the targeted cells can furthermore propagate to non-exposed bystander cells thereby exacerbating the damage. Ca²⁺ signaling is strongly intertwined with ROS signaling and both play crucial roles in cell death. In this review we aimed to review current knowledge on the role of Ca²⁺ and ROS signaling, their effect on cell-cell propagation via connexin-linked mechanisms and the outcome in terms of cell death. In general, photodynamic therapy results in an increased cytosolic Ca²⁺ concentration originating from Ca²⁺ entry or Ca²⁺ release from internal stores. While photodynamic therapy can certainly induce cell death, the outcome depends on the cell type and the photosensitizer used. Connexin channels propagating the Ca²⁺ signal, and presumably regenerating ROS at distance, may play a role in spreading the effect to neighboring non-exposed bystander cells. Given the various cell types and photosensitizers used, there is currently no unified signaling scheme to explain the role of Ca²⁺ and connexins in the responses following photodynamic therapy. This article is part of a Special Issue entitled: Calcium signaling in health, disease and therapy edited by Geert Bultynck and Jan Parys.