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1998;3:228C236. to bring about stress tolerance. Typically, stress tolerance is defined by measuring cell survival after each of 2 consecutive tensions. Initially this trend was called the adaptive response of cells exposed to elevated temperatures because a slight heat shock treatment allows cells to withstand the effect of a second, more intense temp insult, which would normally become lethal. This acquired stress tolerance is recognized as a general and highly conserved strategy that shields cells from many other stress-inducing providers (Samali and Orrenius 1998). It has been previously shown that in sea urchin, embryos degenerate when heated at 35C. On the other hand, they survive and develop if this lethal heat treatment is definitely preceded by heating at 31C (Sconzo et al 1986). Some other nonthermal stress can induce thermotolerance; therefore, for example, sea urchin embryos treated with ethylene glycol-bis(aminoethylether)-tetraacetic acid (EGTA) (which inhibits the bulk protein synthesis and induces the synthesis of the complete set of W-2429 Hsps) survive the normally lethal temp of 35C (Roccheri et al 2000). On the contrary, Zn+ treatment of embryos, which also induces synthesis of Hsps but not the complete repertoire of them, does not inhibit the synthesis of bulk proteins nor induces thermotolerance (Roccheri et al 1988). Thermotolerance can also be accomplished through phosphorylation of some Hsps, such as those of 38C40 kDa, induced by tetradecanoylphorbol-13 acetate (TPA) treatment or warmth shock (Roccheri et al 1995). In the last few years it has become clear CD247 the manifestation of Hsps in response to slight stress, in various biological systems, could induce thermotolerance through inhibition of apoptosis: after phosphorylation, Hsp27 preferentially blocks mitochondrial cytochrome launch, whereas Hsp72 interferes with apoptosomal caspase activation (Arrigo and Orrenius 2001; Beere and Green 2001; Kato et al 2001; Samali et al 2001). Two different transmission transduction cascades may be responsible for phosphorylation of Hsp27: the p38 mitogen-activated protein kinase (MAPk) pathway and the protein kinase C cascade (Kato et al 2001). Numerous forms of cellular stress (hyperosmotic stress, heat shock, deoxyribonucleic acid damage, anysomicin, sodium arsenite, inflammatory cytokines, lipopolysaccharides, ultraviolet irradiation) and also immune response are known to activate p38 stress-activated protein kinase (SAPk). Activation of p38 requires dual phosphorylation of both threonine and tyrosine residues in the activation website. Once triggered, p38 can phosphorylate both nuclear transcription factors, such as ATF2, ELK1, cJun, and cytoplasmic substrates, such as IkB or the small Hsp27 (Kyriakis and Avruch 1996; Han et al 1998; Blanco 2000). The HOG/p38SAPk route is an important osmostress-activated transmission transduction pathway, well conserved in all eukaryotes. In candida, the living of 2 different activation mechanisms, which depend on osmotic conditions, has been shown for this pathway (Vehicle Wuytswinkel et al 2000). We have previously shown that sea urchin embryos, planktonic life of which could depend on swimmer and sensorial ciliary functions, when deciliated by hypertonic shock, develop a stress response that transiently upregulates the manifestation and phosphorylation W-2429 of a 40-kDa stress protein (Casano et al 1998, and unpublished data). In this study, we display that hyperosmotic deciliation of sea urchin embryos induces thermotolerance, activates a phosphorylation pathway, W-2429 and finally activates p38SAPk. MATERIALS AND METHODS Embryo tradition and treatments Adult sea urchins of Mediterranean varieties, collected along the Sicily’s western coast, were used in this study as previously explained (Casano et al 1998). Briefly, eggs were fertilized and embryos, at a concentration of 5000/mL, were cultivated at 18C in Millipore filtered seawater comprising antibiotics under continuous rotation, until the gastrula stage (at earlier stages, sea urchin embryos are unable to respond to stress). Embryo ethnicities were exposed to nonlethal and lethal warmth shock stress and to deciliation as already explained (Roccheri et al 1995; Casano et al 1998). The SB203580 p38 inhibitor was added at concentrations of 30 and 60 M in seawater. Electrophoretic analysis Control and treated embryos were Dounce homogenized in O’Farrel lysis buffer including the protease inhibitor cocktail (total, Mini, ethylenediaminetetraacetic acidCfree protease inhibitor cocktail tablets; Roche, Mannheim, Germany). Equivalent amounts of total proteins (40 g) were analyzed by monodimensional sodium dodecyl sulfate (SDS)C10% polyacrylamide slab gels, as previously explained (Giudice et al 1980). Molecular weights were evaluated by comparing with a set of standard proteins (Rainbow protein molecular excess weight markers; Amersham, W-2429 Buckinghamshire, UK). Western blotting After electrophoresis, the proteins were electroblotted onto nitrocellulose filters (Hybond C or ECL; Amersham) with a semidry apparatus (Novablot; Pharmacia, Peapack, NJ, USA) W-2429 at 0.8 mA/cm2 for 2 hours. The filters were then preincubated for 3 hours with blocking answer (3% bovine serum albumin,.