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,...

Rats were subjected to the standard four-vessel occlusion model of cerebral transient ischaemia (vertebral and carotid arteries) for 15 and 30 min. After a 30 min recirculation period, protein synthesis rate, initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) activities, and the level of phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) were determined in the neocortex region of the brain from sham-operated controls and ischaemic animals. Following reversible cerebral ischaemia, the protein synthesis rate, as measured in a cell-free system, was significantly inhibited (70%) in the ischaemic animals. eIF-2 activity, as measured by its ability to form a ternary complex, also decrease parallel to the decrease in protein synthesis. As eIF-2 activity was assayed in the presence of Mg2+ and GTP-regenerating capacity, the decrease in ternary-complex formation indicated the possible impairment of GEF activity. Since phosphorylated eIF-2 [eIF-2(alpha P)] is a powerful inhibitor of GEF, the levels of phosphorylated eIF-2 alpha were determined, and an increase from 7% phosphorylation in sham control rats to 20% phosphorylation in 15 min and 29% phosphorylation in 30 min in ischaemic rats was observed, providing evidence for a tight correlation of phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Moreover, GEF activity measured in the GDP-exchange assay was in fact inhibited in the ischaemic animals, proving that protein synthesis is impaired by the presence of eIF-2(alpha P), which blocks eIF-2 recycling.

Translational repression induced during reperfusion of the ischaemic brain is significantly attenuated by ischaemic preconditioning. The present work was undertaken to identify the components of the translational machinery involved and to determine whether translational attenuation selectively modifies protein expression patterns during reperfusion. Wistar rats were preconditioned by 5-min sublethal ischaemia and 2 days later, 30-min lethal ischaemia was induced. Several parameters were studied after lethal ischaemia and reperfusion in rats with and without acquired ischaemic tolerance (IT). The phosphorylation pattern of the a subunit of eukaryotic initiation factor 2 (eIF2) in rats with IT was exactly the same as in rats without IT, reaching a peak after 30 min reperfusion and returning to control values within 4 h in both the cortex and hippocampus. The levels of phosphorylated eIF4E-binding protein after lethal ischaemia and eIF4E at 30 min reperfusion were higher in rats with IT, notably in the hippocampus. eIF4G levels diminished slightly after ischaemia and reperfusion, paralleling calpain-mediated a-spectrin proteolysis in rats with and without IT, but they did not show any further decrease after 30 min reperfusion in rats with IT. The phosphorylated levels of eIF4G, phosphatidylinositol 3-kinase-protein B (Akt) and extracellular signal-regulated kinases (ERKs) were very low after lethal ischaemia and increased following reperfusion. Ischaemic preconditioning did not modify the observed changes in eIF4G phosphorylation. All these results support that translation attenuation may occur through multiple targets. The levels of the glucose-regulated protein (78 kDa) remained unchanged in rats with and without IT. Conversely, our data establish a novel finding that ischaemia induces strong translation of growth arrest and DNA damage protein 34 (GADD34) after 4 h of reperfusion. GADD34 protein was slightly up-regulated after preconditioning, besides, as in rats without IT, GADD34 levels underwent a further clear-cut increase during reperfusion, this time as earlier as 30 min and coincident with translation attenuation.

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death. 2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning. 3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons. 4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.

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