Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs.

Mollusc shells are complex organomineral structures, the arrangement and composition depending on the species. Most studies are dedicated to shells composed of an aragonite nacreous and a calcite prismatic layer, so the nacreous prismatic model based on Pinctada and Atrina-Pinna. Here, we studied the micro- and nanostructure, the mineralogy and composition of a nacroprismatic bivalve species: Unio pictorum. The prismatic layer of Unio is aragonite, and the inner structure of the prismatic units strongly differs from those of the calcitic layers. The shape of the prisms varies depending on their growth stage. The first layers of nacre are similar to those of gastropods (columnar nacre), then evolve towards the typical bivalve arrangement (sheet nacre). Na, Sr, Mg, P and S are present in both prisms and nacre. The organic prismatic envelopes are rich in sulphur amino acids, whereas organic sulphate is present within the prisms and the nacreous tablets. P is present as phosphate, probably a mixture of organic and mineral complex. Chemical distribution maps confirm the absence of an organic membrane between the nacre and the prisms. The comparison of the structure, mineralogy and composition of Unio pictorum and different species show the diversity of nacroprismatic shells, and that these features are taxonomically dependent.

Shell repair process in the green ormer Haliotis tuberculata: a histological and microstructural study.

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata ('Oreille de Saint-Pierre') were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297-300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous (i.e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.

Characterization and N-terminal sequencing of a calcium binding protein from the calcareous concretion organic matrix of the terrestrial crustacean Orchestia cavimana.

We extracted proteins from the organic matrix of calcareous concretions, which represents the calcium storage form in a terrestrial crustacean. Electrophoretic analyses of water-soluble organic-matrix proteinaceous components revealed 11 polypeptides, 6 of which are probably glycosylated. Among the unglycosylated proteins, we characterized a 23 kDa polypeptide, with an isoelectric point of 5.5, which is able to bind calcium. Its N-terminal sequence is rich in acidic amino acids (essentially aspartic acid). All these characteristics suggest its involvement in the calcium precipitation process within the successive layers of the organic matrix.

Evidence for the production of an interleukin-like protein by Asterias rubens (axial organ cells) in mixed leukocyte cultures.

Cells from the axial organ of the sea star may be separated into two subpopulations, depending on adherence to nylon wool. The non-adherent cells are morphologically and physiologically similar to Th. lymphocytes and produce an MLR-like reaction in a xenogeneic system involving two different species of sea stars. On the second day of such a MLC, these cells released molecules which were probably glycoproteins and which produced a mitogenic effect on all the axial organ cells. No effect occurred when the supernatant was tested on adherent or non-adherent cells separately, suggesting that a cellular cooperation is necessary in order to produce the response. These soluble mediators produced by T-like cells with a mitogenic effect on lymphocyte-like cells resemble the interleukin proteins produced by vertebrate leukocytes.

In vitro effect of rabbit anti sea star lymphocyte serum on axial organ cells.

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated or not into two populations: adherent (B-like) and non adherent (T-like) to nylon wool. Rabbit anti sea star lymphocyte serum induces the proliferation of axial organ cells. The T-like antiserum stimulates the T-like cells exclusively; the whole axial organ cell antiserum only stimulates the whole axial organ cell population.

Purification of an antibody-like protein from the sea star Asterias rubens (L.).

Cells from sea star (Asterias rubens) axial organs stimulated with trinitrophenyl (TNP) or fluoresceinyl-haptened polyacrylamide beads and subsequently stimulated in vitro with the same antigen produced and released a specific antibody-like protein which induced lysis of haptened sheep erythrocytes in the presence of serum complement. The anti-TNP antibody-like protein isolated by ammonium sulfate precipitation, gel filtration and affinity chromatography exhibited a single precipiting peak after crossed immunoelectrophoresis against rabbit antiserum to partially purified culture supernatant. The anti-TNP antibody-like protein gave a specific affinity precipitate in crossed affino-electrophoresis using a p-nitrobenzoyl-substituted gel. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions evidenced a unique 30-kDa polypeptide chain. According to gel filtration experiments, the molecular weight of the major component isolated by affinity chromatography was about four times higher. Therefore, the antibody-like molecule could be a tetrameric protein devoid of any disulfide bond.

Production of an antibody-like factor in the sea star Asterias rubens: involvement of at least three cellular populations.

Cells from the axial organ of sea stars stimulated in vivo with TNP coupled to polyacrylamide beads and subsequently cultured in vitro were able to produce an antibody-like factor which induced the lysis of mammalian red cells sensitized with TNP. The axial organ cells were fractionated in two populations, adherent and non-adherent to nylon wool. The release of the antibody-like factor required the contact of both populations. When the adherent cells were disrupted by sonication the factor was not produced, but the non-adherent cells could be substituted by their membranes. Destruction by silica of the phagocytic cells present in the adherent population inhibited the production of the factor. The addition of mercaptoethanol to the cultures was essential and did not neutralize the effect of silica. It is concluded that at least three types of cells are involved in the production of the antibody-like factor adherent and non-adherent cells, and phagocytes.

Immunocompetent cells in the starfish Asterias rubens. An ultrastructural study.

Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.

Specific immune response in the sea star Asterias rubens: production of "antibody-like" factors.

Axial organ cells from the sea star (Asterias rubens) inoculated 7 days before with TNP- or FITC-haptenated PAA beads and subsequently stimulated in vitro with the same antigen, produced and released a specific, soluble "antibody-like" substance that induced lysis of haptenated sheep erythrocytes. Fresh normal rabbit or guinea pig serum was essential for the lysis, suggesting the participation of complement components. The factor was produced by the total population of axial organ cells but not by nylon-wool adherent (B-like) or nonadherent (T-like) cells. These results provide further evidence of the existence, in the sea star, of a primitive immune system with characteristics reminiscent of the immune system of vertebrates.

MLR-like reaction between axial organ cells from asterids.

The uptake of [3H]thymidine by axial organ (AO) cells arising from two starfishes and mixed together, in a ratio of 50/50 was studied. Mixed leucocyte reaction (MLR) combinations gave a positive response at the fifth day of culture in about 20% of the tested combination (index: 1.8-6.7). No positive reaction was observed in an allogeneic system. More significant results seem to be obtained with the nonadherent nylon-wool subpopulation cells from the two species, rather than with the whole axial organ cells.

Spontaneous and induced cytotoxicity of axial organ cells from Asterias rubens (Asterid--echinoderm).

Spontaneously occurring cytotoxicity of starfish axial organ cells on target cells has been studied. It appears that in an allogeneic system no cytotoxic phenomenon occurs after 4 or 6 h of culture, whereas spontaneous cytotoxicity toward malignant vertebrate target cells is shown and reaches its maximum at 37 degrees C. In a certain way, the axial organ cell population includes cells, which may be compared with vertebrate natural killer cells. On the other hand, it must also be noted that axial organ cells are able to exert in a few cases, an induced cytotoxicity on MBL2 cells. This phenomenon appears at the end of the fourth day of culture.

Effect of in vivo inoculation of bacteria on the spontaneous cytotoxicity of axial organ cells from Asterias rubens.

In vivo inoculation of heat-killed bacteria in the coelomic cavity of starfish induces an increase of spontaneous cytotoxicity developed by axial organ (an ancestral lymphoid organ) cells, phenomenon demonstrated by an in vitro [51Cr]-release assay. This observation corroborates the existence of a sophisticated immune system in the echinoderms, a first-rate model for phylogenic study of the immune response.

Properties of cell subpopulations of starfish axial organ: in vitro effect of pokeweed mitogen and evidence of lymphokine-like substances.

The in vitro effects of pokeweed mitogen (PWM) on axial organ (AO) cells of the echinoderm starfish Asteria rubens have been studied. PWM stimulates 3H-thymidine incorporation by the whole population of AO cells (index of stimulation 4-5). On the basis of surface adherence to nylon wool the AO cell population can be fractionated into adherent and non-adherent subpopulations. PWM strongly stimulates the non-adherent cells (index 14) but not the adherent cells (index 1.2-1.8). Specific soluble mediators obtained from stimulated non-adherent cells were able to stimulate the whole AO cell population but not the fractionated adherent or non-adherent cells.