Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD ؉ into cyclic ADP-ribose, a potent and universal intracellular Ca 2؉ mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K ؉ channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.A DP-ribosyl cyclase activity is expressed along the phylogenetic tree from unicellular protists (1) to mammals (2). It converts NAD ϩ to cyclic ADP-ribose (cADPR) (3), an intracellular calcium mobilizer that is being increasingly recognized as a pivotal signaling molecule, involved in such diverse functions as cell cycle regulation (1) (protists), oocyte fertilization (4) (invertebrates), insulin secretion (5), and cell proliferation (6) (mammals). The peculiar position of Porifera in the phylogenetic tree [they are the oldest known Metazoa, sharing a common ancestor with all multicellular animals (7)] and the presence of signaling pathways in these organisms (8) prompted us to investigate the presence, and the functional role, of the ADPribosyl cyclase͞cADPR system in marine sponges. Materials and MethodsSponges. Sponges were collected in the Ligurian Sea (Mediterranean Sea), at Gallinara Island (Genova, Italy) at a depth between 35 and 45 m. The temperature was maintained at 14°C during transfer of the animals to the lab. They were kept in an aquarium in natural seawater (SW) at 14°C for 5-7 days before utilization. All procedures involving sponge tissue were performed at 14°C. As the most ancient and simple metazoan animals, sponges lack a defined tissue organization. Cells are embedded in a collagenous matrix, impregnated with siliceous or calcareous spicules, surrounding a complex network of internal canals, through which water circulates. Intact sponge cells (Ϸ8 m diameter) could be easily obtained by gentle squeezing of cleanly cut Axinella polypoides fragments. Cell viability, as checked microscopically, was always Ն95% after mechanical dissociation and after exposure of the cells to heat stress or abscisic acid (ABA).HPLC and Fluorimetric Assays of Enzymatic A...

Collagen is involved in the formation of complex fibrillar networks, providing the structural integrity of tissues. Its low immunogenicity and mechanical properties make this molecule a biomaterial that is extremely suitable for tissue engineering and regenerative medicine (TERM) strategies in human health issues. Here, for the first time, we performed a thorough screening of four different methods to obtain sponge collagenous fibrillar suspensions (FSs) from C. reniformis demosponge, which were then chemically, physically, and biologically characterized, in terms of protein, collagen, and glycosaminoglycans content, viscous properties, biocompatibility, and antioxidant activity. These four FSs were then tested for their capability to generate crosslinked or not thin sponge collagenous membranes (SCMs) that are suitable for TERM purposes. Two types of FSs, of the four tested, were able to generate SCMs, either from crosslinking or not, and showed good mechanical properties, enzymatic degradation resistance, water binding capacity, antioxidant activity, and biocompatibility on both fibroblast and keratinocyte cell cultures. Finally, our results demonstrate that it is possible to adapt the extraction procedure in order to alternatively improve the mechanical properties or the antioxidant performances of the derived biomaterial, depending on the application requirements, thanks to the versatility of C. reniformis extracellular matrix extracts.

Siliceous sponges, one of the few animal groups involved in a biosilicification process, deposit hydrated silica in discrete skeletal elements called spicules. A multidisciplinary analysis of the structural features of the protein axial filaments inside the spicules of a number of marine sponges, belonging to two different classes (Demospongiae and Hexactinellida), is presented, together with a preliminary analysis of the biosilicification process. The study was carried out by a unique combination of techniques: fiber diffraction using synchrotron radiation, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetric (DSC), Fourier transform infrared spectroscopy (FTIR), and molecular modeling. From a phylogenetic point of view, the main result is the structural difference between the dimension and packing of the protein units in the spicule filaments of the Demospongiae and the Hexactinellida species. Models of the protein organization in the spicule axial filaments, consistent with the various experimental evidences, are given. The three different species of demosponges analyzed have similar general structural features, but they differ in the degree of order. The structural information on the spicule axial filaments can help shed some light on the still unknown molecular mechanisms controlling biosilicification.

Sponges are a valuable source of natural compounds and biomaterials for many biotechnological applications. Marine sponges belonging to the order Verongiida are known to contain both chitin and biologically active bromotyrosines. Aplysina archeri (Aplysineidae: Verongiida) is well known to contain bromotyrosines with relevant bioactivity against human and animal diseases. The aim of this study was to develop an express method for the production of naturally prefabricated 3D chitin and bromotyrosine-containing extracts simultaneously. This new method is based on microwave irradiation (MWI) together with stepwise treatment using 1% sodium hydroxide, 20% acetic acid, and 30% hydrogen peroxide. This approach, which takes up to 1 h, made it possible to isolate chitin from the tube-like skeleton of A. archeri and to demonstrate the presence of this biopolymer in this sponge for the first time. Additionally, this procedure does not deacetylate chitin to chitosan and enables the recovery of ready-to-use 3D chitin scaffolds without destruction of the unique tube-like fibrous interconnected structure of the isolated biomaterial. Furthermore, these mechanically stressed fibers still have the capacity for saturation with water, methylene blue dye, crude oil, and blood, which is necessary for the application of such renewable 3D chitinous centimeter-sized scaffolds in diverse technological and biomedical fields.