Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.     

High level transactivation by the ecdysone receptor complex at the core recognition motif.

Ecdysteroid signaling in insects is mediated by the ecdysone receptor complex that is composed of a heterodimer of the ecdysone receptor and Ultraspiracle. The DNA binding specificity plays a critical role of defining the repertoire of target genes that respond to the hormone. We report here the determination of the preferred core recognition motif by a binding site selection procedure. The consensus sequence consists of a perfect palindrome of the heptameric half-site sequence GAGGTCA that is separated by a single A/T base pair. No binding polarity of the ecdysone receptor/Ultraspiracle heterodimer to the core recognition motif was observed. This core motif mediated the highest level of ligand-induced transactivation when compared to a series of synthetic ecdysone response elements and to the natural element of the Drosophila hsp27 gene. This is the first report of a palindromic sequence identified as the highest affinity DNA binding site for a heterodimeric nuclear hormone receptor complex. We further present evidence that the ligand of the ecdysone receptor preferentially drives Ultraspiracle from a homodimer into a heterodimer. This mechanism might contribute additionally to a tight control of target gene expression.     

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.     

Promoter Analysis for Three Types of EUL-Related Rice Lectins in Transgenic Arabidopsis

In this study, the promoter activity for three types of Euonymus-related lectins (EUL) from rice, further referred to as OrysaEULS2, OrysaEULS3, and OrysaEULD1A was analyzed. In silico promoter analyses showed that the EUL promoters from rice contain next to the typical promoter elements some motifs that are considered to be stress-responsive elements. Furthermore, Arabidopsis thaliana plants were transformed with a promoter::β-glucuronidase (GUS) construct for each of the proteins under study. Subsequently, one-insertion homozygous lines were selected and analyzed for GUS activity. Experiments were performed under normal growth conditions or after application of different stress conditions, in particular treatments with 150 mM NaCl, 100 mM mannitol, and 100 μM abscisic acid (ABA) for 24 h. GUS activity was detected with the OrysaEULS3 and OrysaEULD1A promoters especially in the cotyledons and the young true leaves, respectively, but not with the OrysaEULS2 promoter. The activity of OrysaEULS3 and OrysaEULD1A promoters was increased after ABA and mannitol treatments but decreased after NaCl treatment. We hypothesize that the Euonymus-related rice proteins have a role in sensing and responding to external stresses as well as in the growth of the plant.     

Capillary electrophoretic separation and quantification of flavone-O- and C-glycosides in Achillea setacea W. et K

A simple method for the rapid separation and quantification of flavone-O- and C-glycosides in A. setacea W. et K. by capillary zone electrophoresis (CZE) with UV detection is described. Using 25 mM sodium borate with 20% (v/v) of methanol (pH 9.3) as running buffer sufficient separation of the analytes was achieved within 19 min. For the quantitative determination isorhamnetin-3-O-rutinoside was used as internal standard. The method was successfully applied to a rapid characterisation of the flavonoid complex and a precise quantification of the single and total amount of the flavonoids in different samples of A. setacea.