The complete assignment of the proton NMR spectra of the homologous gamma 1-hordothionin and gamma 1-purothionin (47 amino acids, 4 disulfide bridges) from barley and wheat, respectively, has been performed by two-dimensional sequence-specific methods. A total of 299 proton-proton distance constraints for gamma 1-H and 285 for gamma 1-P derived from NOESY spectra have been used to calculate the three-dimensional solution structures. Initial structures have been generated by distance geometry methods and further refined by dynamical simulated annealing calculations. Both proteins show identical secondary and tertiary structure with a well-defined triple-stranded antiparallel beta-sheet (residues 1-6, 31-34, and 39-47), an alpha-helix (residues 16-28), and the corresponding connecting loops. Three disulfide bridges are located in the hydrophobic core holding together the alpha-helix and the beta-sheet and forming a cysteine-stabilized alpha-helical (CSH) motif. Moreover, a clustering of positive charges is observed on the face of the beta-sheet opposite to the helix. The three-dimensional structures of the gamma-thionins differ remarkably from plant alpha- and beta-thionins and crambin. However, they show a higher structural analogy with scorpion toxins and insect defensins which also present the CSH motif.

A new sulfur-rich and basic polypeptide, designated as y-hordothionin, has been isolated from barley endosperm by a semi-preparative purification consisting of extraction with a volatile salt solution followed by highperformance liquid chromatography using a reversed-phase C4 column. The isolated polypeptide was found to be homogeneous by micro-two-dimensional gel electrophoresis in the presence of sodium dodecyl sulfate. The complete primary structure of y-hordothionin was determined by automatic degradation of the intact, Scarboxymethylated and S-pyridylethylated y-hordothionin and fragments obtained by proteolytic cleavage. yHordothionin consists of a single polypeptide chain of 47 amino acids with a calculated molecular mass of 5250 Da and contains four disulfide bridges. y-Hordothionin inhibits translation in cell-free systems derived from mammalian (rabbit reticulocyte, mouse liver) as well as non-mammalian (Artemia embryo) cells, at several levels. At low concentrations (1 -10 pM) the protein seems to affect mainly the polypeptide-chain-initiation process, although it might also act at the elongation level. At higher concentrations (20 -80 pM) this inhibitor induces activation of an eukaryotic polypeptide-chain initiation factor 2 a-subunit (eIF-2a) kinase in hemin-supplemented reticulocyte lysates, as does hemin deficiency. The presence of the disulfide bridges in y-hordothionin appears to be essential for the eIF-2a kinase activation. Based on its similarity at both the structural and functional level with the different genetic variants of thionins (a and p-thionins, from wheat and barley), y-hordothionin is a putative member of the thionin family.Two groups of protein synthesis inhibitors have been described from the endosperm of several Gramineae. Type 1 ribosome-inactivating proteins, which are single-chain basic polypeptides with molecular mass of around 30 kDa and are N-terminally blocked [l], belong to the first group. They have been found in wheat germ and in wheat, barley, rye and corn grains [l -61, as well as in a number of other plant species from different sources [2, 5, 71. An inhibitory effect has been shown with in vitro cell-free systems of protein synthesis from rabbit reticulocyte lysates, Ehrlich ascites cell lysates [2 -51 and with in vivo fungal growths [8]. These molecules inhibit protein synthesis in eucaryotic cells by interfering with the ability of the 60s subunit to bind the elongation factor 2 [2,The second group comprises a family of low-molecularmass proteins of around 5 kDa, rich in basic amino acids and cysteines and named thionins [9-lo]. Thionins are toxic to bacteria [11 -121, certain strains of yeasts [ l l , 131, insect larvae [14] and cultured cells [15-161 and they modify membraneCorrespondence to E. Mendez, Servicio de Endocrinologia, Hospital Ramon y Cajal, E-28034 Madrid, SpainAbbreviations. cIF-24 the a subunit of eukaryotic polypeptidechain initiation factor 2; HCI, heme-controlled translational inhibitor (an eIF-2cr kinase); GSSG, oxidized glutathione; Pth,...

Two homologous sulfur-rich basic polypeptides form wheat endosperm, so-called y,-purothionin and y,-purothionin, are described. Purification involves extraction with volatile solvents and ammonium bicarbonate fractionation followed by reversed-phase high-performance liquid chromatography. The complete primary structure of these two polypeptides has been determined by automatic degradation of the intact, S-carboxymethylated y-purothionins and peptides obtained by enzymatic cleavage. y,-Purothionin and y,-purothionin consist of 47 amino acids with an assessed molecular weight of 5239 and 5151 Da, respectively and 8 cysteines organ&J in 4 disulfide bridges. They present a high degree of homology among themselves (89% of identity) and are the first two thionin-like polypeptides, so-called y-thionins, described from wheat endospemt.Wheat thionin; Amino acid sequence; y-Purothionin

Fernández-Torquemada, Y., Díaz-Valdés, M., Colilla, F., Luna, B., Sánchez-Lizaso, J. L., and Ramos-Esplá, A. A. 2008. Descriptors from Posidonia oceanica (L.) Delile meadows in coastal waters of Valencia, Spain, in the context of the EU Water Framework Directive. – ICES Journal of Marine Science, 65: 1492–1497. Evaluations are provided of 21 descriptors of Posidonia oceanica meadows along the coast of Valencia, Spain, with a view to using these in implementing the European Water Framework Directive (WFD). The descriptors selected are known to respond to a variety of anthropogenic disturbances. Data were collected at 17 locations during three consecutive years. A principal components analysis was used to classify the ecological status of each locality according to five classes as prescribed by the WFD. To identify the descriptors that contributed most to similarity among localities within each class and to dissimilarity between adjacent classes, a similarity percentage analysis was performed. We also correlated the descriptors with an independent set of indicators for various types of anthropogenic pressures on the water bodies associated with the different localities. The descriptors providing the most consistent information on status as well as demonstrating a significant relationship with estimated pressures were: shoot density, shoot foliar surface, dead-matte cover, meadow cover, herbivore pressure, rhizome baring/burial, foliar necrosis, percentage of plagiotropic rhizomes, and leaf-epiphyte biomass.

Six chloroform/mcthanol-solubl¢ proteins from oat endosperm (Avena saliva) have been isolated and characterized by a purification procedure based on extraction with volatile solvents, followed by reversed-phase high performan~ liquid chromatography. Three of these proteins, with an assessed mol~ular weight of 25,000, 27,000 and 32,000 Da, respectively, have been identified by immunoblotting using ¢oeliac sere, as the major co¢liac serum leA-binding components of oat endosperm. The N-terminal amino acid sequence of these proteins indicates that they correspond to oh., 74, and 7a avenins, respectively. We have tentatively named them 'coeliac immunoreactive proteins'. Another chloroform/methanol oat component shows weak g-amylase inhibitory activity and exhibits strong homology (60% identity) at the N-tgrmin~ with the g-amylase inhibitor from ragi (Eleusilze coracana).