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The sea urchin embryo toxicity test is classically used to assess the noxious effects of contaminated marine waters and sediments. In Italian guidelines on quality of dredged sediments, the standard toxicity criteria used for this assay are based on a single endpoint at 48 hours of development, corresponding to the pluteus stage. Different typologies of abnormalities, including those which occur at earlier stages, are not categorized, thus preventing the evaluation of the actual teratogenic hazards. A new integrative toxicity index has been developed in this study based on the analysis of two developmental stages, at 24 and 48h post-fertilization, and the differentiation between development delays and germ layers impairments: the new toxicity index is calculated by integrating the frequency of abnormal embryos with the severity of such abnormalities. When tested on dredged sediments, the evaluation of increasing levels of toxicity affecting embryonic outcomes enhanced the capability to discriminate different samples, appearing particularly relevant to validate the sea urchin embryo toxicity assay, and supporting its utility in practical applications such as the sediments classification in harbor areas.

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In the marine environment, manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. In earlier reports we found that the exposure of Paracentrotus lividus sea urchin embryos to manganese produced phenotypes with no skeleton. In addition, manganese interfered with calcium uptake, perturbed extracellular signal-regulated kinase (ERK) signaling, affected the expression of skeletogenic genes, and caused an increase of the hsc70 and hsc60 protein levels. Here, we extended our studies focusing on the temporal activation of the p38 mitogen-activated protein kinase (p38 MAPK) and the proteolytic activity of metalloproteinases (MMPs). We found that manganese affects the stage-dependent dynamics of p38 MAPK activation and inhibits the total gelatin-auto-cleaving activity of MMPs, with the exclusion of the 90-85 kDa and 68-58 kDa MMPs, whose levels remain high all throughout development. Our findings correlate, for the first time to our knowledge, an altered activation pattern of the p38 MAPK with an aberrant MMP proteolytic activity in the sea urchin embryo.

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The potential toxicity of stannum dioxide (SnO2), cerium dioxide (CeO2) and iron oxide (Fe3O4) nanoparticles (NPs) in the marine environment was investigated using the sea urchin, Paracentrotus lividus, as an in vivo model. We found that 5 days after force-feeding of NPs in aqueous solutions, the three NPs presented different toxicity degrees, depending on the considered biomarkers. We examined: 1) the presence of the NPs in the coelomic fluid and the uptake into the immune cells (coelomocytes); 2) the cholinesterase activity and the expression of the stress-related proteins HSC70 and GRP78; 3) the morphological changes affecting cellular compartments, such as the endoplasmic reticulum (ER) and lysosomes. By Environmental Scanning Electron Microscope (ESEM) analysis, coupled with Energy Dispersive X-ray Spectroscopy (EDS) we found that NPs were uptaken inside coelomocytes. The cholinesterases activity, a well known marker of blood intoxication in vertebrates, was greatly reduced in specimens exposed to NPs. We found that levels of stress proteins were down-regulated, matching the observed ER and lysosomes morphological alterations. In conclusion, this is the first study which utilizes the sea urchin as a model organism for biomonitoring the biological impact of NPs and supports the efficacy of the selected biomarkers.

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The common sea star Asterias rubens represents a key-species of the North-Eastern Atlantic macro benthic community. The cells of their immune system, known as coelomocytes, are the first line of defence against environmental hazards. Here, we report the results of investigations on the immune cells response of sea stars exposed to marine environmental pollution for long periods. We show that levels of the heat shock cognate protein 70 (HSC70) in coelomocytes from A. rubens, which were collected during a field study in the SÇ¿rfjord (North Sea, SW coast of Norway) along a contamination gradient, are directly associated with the long-term accumulation of Cd, Cu heavy metals exclusively in the tegument. Conversely, Pb and Zn accumulation in the tegument did not relate to HSC70 levels and none of the metals were found accumulated in the pyloric coeca. In addition the coelomocytes from A. rubens, collected in high and low metal impacted stations were examined by a proteomic approach using two-dimensional electrophoresis (2DE). By comparison of the proteomic maps, we observed that 31 protein spots differed in their relative abundance, indicating a gene expression response to the metal mixture exposure. All together, our results confirm that the echinoderm immune cells are a suitable model for the assessment of long-term exposure to environmental pollution, moreover that the increased level of HSC70 can be considered a signal of an acquired tolerance within a large spectrum of protein profile changes occurring in response to metal contamination.

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Studies on the effects of a variety of exogenous and anthropogenic environmental factors, including endocrine disruptors, heavy metals, UV light, high temperature, and others, on marine organisms have been presented at the 2nd Bilateral Seminar Italy-Japan held in November 2006. Reports were discussed in order to reveal the current situation of marine ecosystems, aiming at evaluation and prediction of environmental risks.

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The aim of the present study was to investigate on the suitability of the sea urchin as a sentinel organism for the assessment of the macro-zoobenthos health state in bio-monitoring programmes. A field study was carried out during two oceanographic campaigns using immuno-competent cells, the coelomocytes, from sea urchins living in a marine protected area. In particular, coelomocytes subpopulations ratio and heat shock protein 70 (HSC70) levels were measured in specimens of Paracentrotus lividus (Lamark, 1816) collected in two sampling sites, namely Pianosa and Caprara Islands, both belonging to the Tremiti Island Marine Protected Area (MPA) in the Southern Adriatic Sea, Italy. By density gradients separation performed on board the Astrea boat, we found an evident increase in red amoebocytes, a subpopulation increasing upon stress, in those specimens collected around Pianosa (strictly protected area with no human activities allowed), unlike those collected around Caprara (low restrictions for human activities). Likewise, we found higher HSC70 protein levels in the low impacted site (Pianosa) by Western blots on total coelomocyte lysates. The apparent paradox could be explained by the presence in the Pianosa sampling area of contaminating remains from Second World War conventional ammunitions and a merchant boat wreck. Metal determination performed using sea urchin gonads by inductively coupled plasma atomic emission spectrometry (ICP-AES) revealed higher Fe and lower Zn levels around Pianosa with respect to Caprara, in accordance with the persistent contaminating metal sources, and thus calling for remediation measures. Taken all together, our results confirm the feasibility of using sea urchin coelomocytes as biosensors of environmental stress.

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In this chapter, we summarise fundamental findings concerning echinoderms as well as research interests on this phylum for biomedical and evolutionary studies. We discuss how current knowledge of echinoderm biology, in particular of the sea urchin system, can shed light on the understanding of important biological phenomena and in dissecting them at the molecular level. The general principles of sea urchin embryo development are summarised, mainly focusing on cell communication and interactions, with particular attention to the cell-extracellular matrix and cell-cell adhesion molecules and related proteins. Our purpose is not to review all the work done over the years in the field of cellular interaction in echinoderms. On the contrary, we will rather focus on a few arguments in an effort to re-examine some ideas and concepts, with the aim of promoting discussion in this rapidly growing field and opening new routes for research on innovative therapeutic tools.

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Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stress. Only recently, coelomocytes have been used for pollution studies with the aim of introducing a new biosensor for detection of stress at both cellular and molecular levels, as sentinel of sea health. In this chapter, we briefly review the important features of these valuable cells and describe studies on their use in the laboratory and in the field for the assessment of chemical and physical pollution of the sea.

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The depletion of the stratospheric ozone layer and the resulting increase in hazardous ultraviolet-B (UV-B) radiation reaching the Earth are of major concern not only for terrestrial but also for aquatic organisms. UV-B is able to penetrate clear water to ecologically significant depths. This chapter deals with the effects of UV radiation on DNA integrity in marine benthic organisms, in particular sea urchins in comparison to other marine invertebrates (sponges and corals). These animals cannot escape the damaging effects of UV-B radiation and may be additionally exposed to pollution from natural or anthropogenic sources. Besides eggs and larvae that lack a protective epidermal layer and are particularly prone to the damaging effects of UV radiation, coelomocytes from the sea urchin Paracentrotus lividus were used as a "cellular sensor" to analyse the effects on DNA caused by UV-B, heavy metals (cadmium), and their combined actions. From our data we conclude that sea urchin coelomocytes as well as cells from other marine invertebrates are useful bioindicators of UV-B and heavy metal stress, responding to these stressors with different extents of DNA damage.

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Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.

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Coelomocytes are considered to be immune effectors of sea urchins. Subpopulations of coelomocytes can be purified from a total cell suspension. The proportion of each cell type can vary not only among species, but also between individuals of the same species, according to their size and physiological conditions. We tested the hypothesis that coelomocytes play a role in defense mechanisms activated by adverse external conditions. Total coelomocytes from control and stressed (temperature, pollution, and injuries) sea urchins were analyzed for their expression of the 70 kDa heat shock protein (hsp70), a well recognized stress marker. Further analysis was performed by separation of coelomocytes into subpopulations by step gradients. We demonstrated that sea urchin coelomocytes respond to temperature shock and to polluted seawater by the upregulation of hsp70. Among coelomocytes certain cells, known as red spherula cells, showed a great increase in number in animals collected from polluted seawaters or subjected to "accidental" injury. The present study confirms the immunological function of sea urchin coelomocytes, as indicated by the upregulation of the hsp70 molecular marker, and suggests that sea urchin coelomocytes can be utilized as sensitive bio-indicators of environmental stress.

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The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.

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Paracentrotus lividus sea urchin nectin (Pl-nectin) is an extracellular matrix (ECM) protein of the sea urchin embryo on the apical surface of the ectoderm and has been shown to be an adhesive substrate for embryonic cells. A monoclonal antibody (McAb) to Pl-nectin was generated that inhibits the adhesion of blastula cells to Pl-nectin-coated substrates in an in vitro functional assay. To examine for possible in vivo functions of Pl-nectin, Fab fragments (Fabs) of Pl-nectin McAb were added to early blastulae. Ingression of primary mesenchyme cells was not affected by Fabs. As control embryos reached the pluteus stage, treated embryos showed a severe inhibition of skeletal elongation and patterning. When the Fabs were injected directly into the blastocoel, even at higher concentration than was applied externally, skeletogenesis was normal. Therefore, the effect of the antibody on spiculogenesis was indirect. The treatment was partially reversible as embryos eventually seemed to recover and elongate spicules, although with an incorrect patterning. Migration of pigment cells was also affected by the Fabs, since they did not disperse throughout the ectoderm but remained clustered in ectopic areas. In contrast, the development of endoderm structures was not affected. Our results indicate that in the sea urchin embryo the appropriate contact of ectodermal cells with outer ECM components is essential for the correct morphogenesis of inner mesodermal structures.

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Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex from Paracentrotus lividus sea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180-160 kDa and a lower band of 80 kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.

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The neural cell adhesion molecule (N-CAM) plays a significant role in the development of the nervous system. Three different isoforms of the molecule have been described, with molecular masses of 180, 140 and 120 kDa, whose differential expression in neurons seems to be related to their state of differentiation. We took advantage of the use of the human neuroblastoma cell line LAN-5, which can be differentiated in vitro by retinoic acid (RA) into neuronal cells, for studying the expression of N-CAM isoforms, and their polysialic acid (PSA) content, at the protein and mRNA levels. Anti-N-CAM polyclonal antibodies recognizing all the N-CAM isoforms and a monoclonal antibody recognizing PSA were used in Western blot experiments with extracts from undifferentiated and RA-differentiated cells. We found that undifferentiated cells express very little of the 180 kDa N-CAM isoform and a large amount of the 140 kDa isoform. A 4-fold increase in the expression of the 180 kDa N-CAM isoform was obtained when LAN-5 cells were differentiated by RA for 8 days, whereas a 1.8-fold increase in the expression of the 140 kDa N-CAM isoform was observed upon differentiation. Similarly, the levels of the 7.4 kb mRNA coding for N-CAM 180 kDa, determined by Northern blot analysis, were barely detectable in undifferentiated cells, and showed a 3.8-fold increase upon differentiation. By contrast, only a 1.3-fold increase in the 6.7 kb mRNA, coding for the 140 kDa N-CAM isoform, was observed. N-CAM was always found in its polysialylated form in both undifferentiated and RA-differentiated cells. This indicates that, in LAN-5 cells, the expression and activity of the polysialytransferase enzyme precedes the acquisition of a neuronal phenotype.

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The glycosyl-phosphatidylinositol (GPI)-anchored F3 molecule, a member of the Ig superfamily made up of Ig and FNIII-like domains, is involved in cell-cell adhesion, neuronal pathfinding and fasciculation. Little is known about the mechanism(s) that governs the F3-mediated cell-cell recognition. In particular, it is not known whether F3 transduces signals across the membrane. Here we show that in F3-transfected CHO cells (1A cells) an increase in tyrosine phosphorylation occurs during F3-mediated aggregation. Moreover, under aggregation conditions F3 immunoprecipitated from 32P-metabolically labeled 1A cells associated with three major phosphorylated proteins. Interestingly, genistein inhibited the F3-mediated aggregation. Increased tyrosine phosphorylation was also observed using antibody-mediated F3-cross-linking. Furthermore, F3 expressed both in 1A cells and in post-natal mouse cerebellum forms non-covalent soluble complexes with protein tyrosine kinase(s). In cerebellum the F3-associated kinase was identified as fyn. By contrast, a truncated F3 protein, expressed in CHO cells, from which all the FN type III repeats have been deleted, does not associate with a kinase. Cross-linking of the F3-truncated form does not induce modulation of tyrosine phosphorylation. Taken together these data demonstrate that F3 is a molecule that transduces signals through both association with protein tyrosine kinase and modulation of protein tyrosine phosphorylation. The presence of FN type III domains is essential for the activation of the intracellular signaling pathway.

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Sea urchin embryos of the species Paracentrotus lividus were treated continuously with different concentrations of all-trans retinoic acid (RA) or dimethylsulfoxide (DMSO) at different developmental stages. A delay in embryonic development was observed when embryos were cultured in the presence of 2 x 10(-5) M RA, between 1 and 12 hours of development. Hence, at 48 hours of development, while control embryos had reached the pluteus stage, RA-treated embryos were at the prism stage. At 72 hours of development RA-treated embryos recovered and continued normal development reaching the pluteus stage. No effect was observed when treatment was performed before 1 hour or after 12 hours of development. DMSO treatment had no effect on normal sea urchin embryo development, although we observed that pigment cells, clearly visible at the pluteus stage, become visible earlier with respect to control embryos. This report confirms the advantages that the sea urchin embryo offers for the study of problems in cellular and developmental biology.

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Fibronectin-like proteins were purified from ovaries of the sea urchin species, Paracentrotus lividus (PI), Sphaerechinus granularis (Sg), Arbacia lixula (Al), Pseudocentrotus depressus (Pd), and Anthocidaris crassispina (Ac), by gelatin-Sepharose affinity chromatography. The major component had a molecular mass of 180 kDa and was eluted by 1 M NaCl or 8 M urea, depending on the species used. By substrate adhesion assay, we tested the biological activity of the 180 kDa protein purified from Paracentrotus lividus (P1-180K) and showed that it promotes the adhesion of homologous embryonic cells to the substrate. An antiserum, developed against Temnopleurus hardwickii fibronectin-like protein (Th-180K), was used in Western blots of the proteins purified from the five species. The antibody cross-reacted with Pl-180K, Pd-180K and Ac-180K. A peptide map of P1-180K, obtained by V8 protease partial digestion, was compared with those obtained from the other four proteins and showed an homology between 40 and 56%. This report confirms that fibronectin-like proteins can be purified from sea urchins on the basis of their binding to gelatin-Sepharose; the proteins differ for their binding affinity to gelatin and share different epitopes, suggesting that they are members of a sea urchin fibronectin super family.

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Sea urchin vitellogenin is a high molecular weight glycoprotein, which is the precursor of the major yolk protein present in the unfertilized egg. Vitellogenin processing into the major yolk protein and its further enzymatic cleavage during sea urchin embryonic development, has been extensively described, and the adhesive properties of the processed molecule have been studied. The function of vitellogenin in the adult, where it has been found in the coelomic fluid of both male and female individuals, is still unknown, although its role on promoting the adhesion of embryonic cells has been shown. In this report we describe the detection of vitellogenin in lysates of whole circulating coelomocytes of both male and female sea urchins of the species Paracentrotus lividus. By metrizoic acid gradients we purified total coelomocytes into six subpopulations that were tested for the occurrence of the molecule using vitellogenin-specific polyclonal antibodies. We detected vitellogenin only in the coelomocyte subpopulation called colorless spherule cells, packed in kidney-shaped granules located around the nucleus. We also showed that coelomocytes respond to stress conditions by discharging vitellogenin into the medium. This result together with previous observations on the adhesive properties of the molecule suggest a role for vitellogenin in the clotting phenomenon occurring after host invasion.

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