The mechanically adaptive connective tissue of echinoderms: its potential for bio-innovation in applied technology and ecology.

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

Mechanical adaptability of a sponge extracellular matrix: Evidence for cellular control of mesohyl stiffness in Chondrosia reniformis Nardo.

The marine sponge Chondrosia reniformis Nardo consists largely of a collagenous tissue, the mesohyl, which confers a cartilaginous consistency on the whole animal. This investigation was prompted by the incidental observation that, despite a paucity of potentially contractile elements in the mesohyl, intact C. reniformis stiffen noticeably when touched. By measuring the deflection under gravity of beam-shaped tissue samples, it was demonstrated that the flexural stiffness of the mesohyl is altered by treatments that influence cellular activities, including [Ca2+] manipulation, inorganic and organic calcium channel-blockers and cell membrane disrupters, and that it is also sensitive to extracts of C. reniformis tissue that have been repeatedly frozen then thawed. Since the membrane disrupters and tissue extracts cause marked stiffening of mesohyl samples, it is hypothesised that cells in the mesohyl store a stiffening factor and that the physiologically controlled release of this factor is responsible for the touch-induced stiffening of intact animals.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor-beta (TGF-beta) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF-beta-like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF-beta-like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non-neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF-beta-like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Regenerative response and endocrine disrupters in crinoid echinoderms: arm regeneration in Antedon mediterranea after experimental exposure to polychlorinated biphenyls.

Regenerative phenomena, which have the advantage of reproducing developmental processes in the adult organism, are very sensitive to environmental stress and represent stages that can be monitored for damage at the whole-organism, cellular and molecular levels. Some persistent and ubiquitous pollutants, which can affect the natural environment because of their bioaccumulation in organisms, exert their effects by acting as 'endocrine disrupters'. In this respect, they can cause dysfunction in steroid hormone production/metabolism and activity by their dramatic effects on gene expression, reproductive competence and growth. The aim of our present research was to assess the impact of such compounds on adult echinoderm reproductive physiology with particular reference to regeneration potential. It is known that vertebrate-type steroids are synthesized by echinoderms and play a role in the control of growth and reproduction. Our experimental model is the crinoid Antedon mediterranea, selected on the basis of its previously explored regenerative capabilities at the level of the arms. The regeneration response, analyzed at the tissue and cellular levels using both light and electron microscopy and immunocytochemistry, was employed to monitor the effects of exposure to persistent endocrine disrupter micropollutants such as polychlorinated biphenyls (PCBs) by means of laboratory tests performed under controlled conditions in terms of environmental variables and contamination levels. Our results indicate that exposure to endocrine disrupter compounds such as PCBs can induce anomalies in regeneration times, morphology and developmental mechanisms that can be interpreted in the light of significant dysfunctions in the endocrine mechanisms controlling regenerative development.

Growth factors, heat-shock proteins and regeneration in echinoderms.

The study of regeneration in armed echinoderm species, including crinoids, ophiuroids and asteroids, is attracting increasing attention. Recent interest has focused on the presence and potential role of growth factors, including members of the nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) families, in the regenerative process and their possible relationship to the normal developmental (ontogenetic) regulatory cascade. In addition, the expression patterns of the heat-shock family of stress proteins (Hsps) during regeneration are also important. Their role forms part of a normal stress response to the trauma of autotomy in combination with a putative function in tissue remodelling and associated protein turnover during regeneration. The temporal dynamics of the stress response may also be strongly indicative of environmentally adaptive pressures operating on these systems.

Changes in ubiquitin conjugates and Hsp72 levels during arm regeneration in echinoderms.

All organisms show a common defensive mechanism that results in the expression of conserved heat shock proteins (Hsps). These proteins function in a wide range of stressful conditions. We have monitored their levels in species of regenerating echinoderms with different mechanisms of regeneration and from different geographical locations. The effect of an artificial higher temperature on expression of Hsps was also studied. Two stress proteins (Hsp72 and ubiquitin) that are important in processes such as development and protein degradation were investigated. Using Western blot analysis and immunocytochemistry, we found significant changes in the level (Hsp72) and pattern of conjugation (ubiquitin) that corresponded with the repair phase (early regenerative stages) and with the later growth and regeneration of new tissues. Animals from the intertidal environment showed a distinctly sustained expression pattern of Hsp72 compared with benthic animals which suggests a functionally adaptative and dynamic stress response program.

Microscopic overview of crinoid regeneration.

Crinoids are well known for their striking regenerative potential and can rapidly and completely regenerate arms lost following self-induced or traumatic amputation. Thus they provide a valuable experimental model for investigation of the regenerative process from the macroscopic to the molecular level. In these last years we have studied in detail the overall process of arm regeneration in the comatulid Antedon mediterranea. This phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotential, actively proliferating cells in the presence of presumptive regulatory factors. The overall process can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as cell migration and proliferation, intervention of stem cells and/or dedifferentiated cells, contribution of putative growth factors, particularly in terms of specific neurally derived factors, and mechanisms of pattern formation. This article focuses on the main aspects of the phenomenon and gives a brief account of the most recent and relevant results. Our approach employs classical methods of light (LM) and electron (TEM and SEM) microscopy, immunocytochemistry, and histofluorescence on experimentally induced arm regenerations of standard or abnormal type obtained in significantly different experimental conditions, including extreme mutilations (explants) or exposure to pseudo-estrogenic environmental contamination.

Identification of particular epithelial areas and cells that transport polypeptide-coated nanoparticles in the nasal respiratory mucosa of the rabbit.

The active transcytosis of many different polypeptides (either presented free or adsorbed on latex nanoparticles), found in the respiratory mucosa of the upper nasal concha, has previously been shown to be proportional to the total volume of the lymphoid aggregates present in the tissue. By combining the use of fluorescent nanoparticles, flux measurements, confocal and scanning electron microscopy and conventional histology, it is shown in this paper that: (i) the areas of epithelium overlying lymphoid aggregates are the only transporting polypeptides; (ii) the respiratory epithelium in these areas consists mainly of non-ciliated microvillar cells, with numerous ciliated cells and rare mucous goblet cells at the periphery of the area only; (iii) non-ciliated microvillar cells are distinguishable in cells with well developed finger-like microvilli and cells with an irregularly pleated apical membrane, similar to that of intestinal and bronchial antigen-sampling M-cells; (iv) groups of polypeptide-coated nanospheres are found bound to this latter type of cells, demonstrating that these are the transporting cells, detected at the first stage of the transcytotic cycle.

Cellular and molecular mechanisms of arm regeneration in crinoid echinoderms: the potential of arm explants.

Crinoid echinoderms can provide a valuable experimental model for studying all aspects of regenerative processes from molecular to macroscopic level. Recently we carried out a detailed study into the overall process of arm regeneration in the crinoid Antedon mediterranea and provided an interpretation of its basic mechanisms. However, the problem of the subsequent fate of the amputated arm segment (explant) once isolated from the animal body and of its possible regenerative potential have never been investigated before. The arm explant in fact represents a simplified and controlled regenerating system which may be very useful in regeneration experiments by providing a valuable test of our hypotheses in terms of mechanisms and processes. In the present study we carried out a comprehensive analysis of double-amputated arm explants (i.e. explants reamputated at their distal end immediately after the first proximal amputation) subjected to the same experimental conditions as the regenerating donor animals. Our results showed that the explants undergo similar regenerative processes but with some significant differences to those mechanisms described for normal regenerating arms. For example, whilst the proximal-distal axis of arm growth is maintained, there are differences in terms of the recruitment of cells which contribute to the regenerating tissue. As with normal regenerating arms, the present work focuses on (1) timing and modality of regeneration in the explant; (2) proliferation, migration and contribution of undifferentiated and/or dedifferentiated/transdifferentiated cells; (3) putative role of neural growth factors. These problems were addressed by employing a combination of conventional microscopy and immunocytochemistry. Comparison between arm explants and regenerating arms of normal donor adults indicates an extraordinary potential and regenerative autonomy of crinoid tissues and the cellular plasticity of the phenomenon.

Leucine transport in Xenopus laevis oocytes: functional and morphological analysis of different defolliculation procedures.

L-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, L-leucine uptake was reduced by 67.5% +/- 5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5% +/- 6.4 after mechanical defolliculation. The Na(+)-dependent uptake of 0.1 mM L-leucine was 18.6 +/- 4.6 pmol oocyte-1 40 min-1 in folliculated oocytes and 5.6 +/- 1.9 in collagenase defolliculated oocytes (means +/- SE). L-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled L-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different L-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific L-leucine binding to membranes. L-leucine kinetics showed that the L-leucine Vmax and Km values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The Vmax and Km values of Na(+)-dependent L-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16 +/- 1.5 pmol oocyte-1 40 min-1 and 57 +/- 21 mumol (mean +/- SD). The Na(+)-activation curve of 0.1 mM L-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of L-leucine Vmax, and microscopic observation showing recovery of the microvillar apparatus.

Pattern of bromodeoxyuridine incorporation in the advanced stages of arm regeneration in the feather star Antedon mediterranea.

The overall process of arm regeneration in the crinoid Antedon mediterranea is a typical epimorphic process (blastemal regeneration). This can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase. The crucial problem of the identification of cell lineages responsible for both repair and regenerative processes has been approached by monitoring cell proliferation during the advanced regenerative phase using light-microscopic and ultrastructural immunocytochemical methods to detect the incorporation of the thymidine analogue bromodeoxyuridine (BrdU) into regenerating tissues. Various treatment protocols and BrdU incubation times have been employed and provided information not only on the sources, sites of proliferation, and recruitment times of the new cells, but also on the cell lineage involved and subsequent fate (differentiation and/or migration) of the labelled cells. Our results are consistent with the following conclusions: (1) The arm regeneration process is due to a massive intervention of active proliferating cells identifiable as migratory, morphologically undifferentiated cells (amoebocytes and coelomocytes). (2) The preferential proliferation sites of these cells are the terminal blastema, the coelomic epithelium, and the brachial nerve of both the regenerating arm and the stump, even far from the amputation. (3) The two main cell components contributing to the regenerate have different origins: the blastemal cells and all the cell lineages derived from the amoebocytes; the coelomic cells from the migratory coelomocytes, in their turn derived from proliferation of the coelomic epithelium. (4) The blastemal regeneration of Antedon is due to a combined recruitment of pluripotent elements, implying the intervention of presumptive stem cells (amoebocytes) and the transdifferentiation/dedifferentiation of differentiated elements of the coelomic epithelium.

Dynamic changes in the state of actin polymerization in human alveolar cells exposed to the oxidant agent paraquat.

To investigate the role of dynamic changes in actin cytoskeleton in cellular response to oxidative stress, we have analyzed the state of actin polymerization and synthesis in human alveolar cells exposed to paraquat, an oxidant agent. Cellular content of monomeric actin was assayed by DNase I inhibition. It decreased significantly in treated cells and depended on paraquat concentration. Paraquat treatment of cells caused an increase of the filamentous pool of actin and a parallel decrease of the monomeric one. Such shift was shown to be irreversible. SDS-PAGE of cytoskeletal fractions was performed under reducing and non-reducing conditions. No cross-linking of actin monomers to form large aggregates appeared to be related to the observed paraquat-induced increase of the filamentous actin pool. Morphological analyses by indirect immunofluorescence and ultrastructural examination confirmed the presence of microfilaments in treated cells. Conventional bundles of filaments were not observed, but numerous single filaments appeared dispersed within the cytoplasm. Pulse-chase experiments showed a strong increase of de novo synthesis of actin in treated cells, whereas actin degradation rate remained unaffected. In conclusion, the different approaches lead to a concordant picture of cellular response to oxidant stress at the level of the actin filament system. Actin pools are modified: the overall number of filaments increases, whereas the monomeric species decreases. As a result of the shift of actin from the monomeric pool to the filamentous one, the de novo synthesis of actin is increased.