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Conventional synthetic methodologies mostly bar access to the fabrication of innovative silica- or metal oxide-based materials with an expanded property spectrum. However, nature represents an inexhaustible source of inspiration for the synthesis of multifunctional materials with tailored attributes. Particularly the low-energy catalytic pathways of poriferan biosilicification, with the enzyme silicatein at their heart, have been inspiring recent biotechnological approaches.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to ortho-phosphate used for bone hydroxyapatite formation. Here we show that the flavone-loaded Ca-polyP microparticles are readily taken up by phOSB, resulting in the accumulation of polyP around the nuclei and the formation of intracellular vesicles containing the ALP. In addition, we demonstrate that baicalin/baicalein causes a rise of the intracellular calcium [Ca[Formula: see text]]i a level which markedly is augmented after encapsulation into Ca-polyP, through activation of the phospholipase C. Moreover, both flavones, either alone or associated with Ca-polyP microparticles, upregulate the expression of the osteoblast calcium efflux channel, the plasma membrane Ca[Formula: see text]-ATPase (PMCA), while the expression of ALP, which promotes bone mineralization, is induced by Ca-polyP and by the flavones only if present in the Ca-polyP-microparticle-associated form. As a result, the extent of bone mineralization is markedly enhanced. Based on the two-armed activating function, new applications of baicalin/baicalein as a component of nutriceuticals for osteoporosis prevention or bone implants can be envisaged.

Impaired regeneration of the corneal epithelium, as found in many ocular surface diseases, is a major clinical problem in ophthalmology. We hypothesized that corneal epithelial regeneration can be promoted by the physiological, energy-delivering as well as “morphogenetically active” polymer, inorganic polyphosphate (polyP). Corneal limbal explants (diameter, 4 mm) were cultivated on collagen-coated well plates in the absence or presence of polyP (chain length, ∼40 Pi units; 50 μg ml−1) or human platelet lysate (hp-lysate; 5% v/v). Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning electron microscopy (ESEM). Cell growth/viability of hCECs was assessed by XTT assay. The expression of SDF-1 was quantitated by qRT-PCR. Exposure to hp-lysate (also containing polyP) increased cell migration already at day 1. Even stronger was the effect of polyP. This effect was blocked by a mast cell serine protease. The formation of cell multilayers was enhanced by hp-lysate or even more by polyP. ESEM revealed continuous cell junctions and prominent microvilli on the surface of adjacent cells exposed to polyP; those structures were only rarely seen in the controls. The hp-lysate and, more potently, polyP increased the proliferation of hCECs, as well as SDF-1 expression. The findings indicate the potential usefulness of the natural polymer, polyP, for topical treatment of corneal epithelial defects. Future studies are directed to develop suitable formulations of polyP, such as biomimetic polyP nano/microparticles showing an adjustable release kinetics.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a recently discovered family of neurotrophic factors. MANF can be secreted but is generally resident within the endoplasmic reticulum (ER) in neuronal and non-neuronal cells, where it is involved in the ER stress response with pro-survival effects. Here we report the discovery of the MANF homolog SDMANF in the sponge Suberites domuncula. The basal positioning of sponges (phylum Porifera) in the animal tree of life offers a unique vantage point on the early evolution of the metazoan-specific genetic toolkit and molecular pathways. Since sponges lack a conventional nervous system, SDMANF presents an enticing opportunity to investigate the evolutionary ancient role of these neurotrophic factors. SDMANF shares considerable sequence similarity with its metazoan homologs. It also comprises a putative protein binding domain with sequence similarities to the Bcl-2 family of apoptotic regulators. In Suberites, SDMANF is expressed in the vicinity of bacteriocytes, where it co-localizes with the toll-like receptor SDTLR. In transfected human cells, SDMANF was detected in both the organelle protein fraction and the cell culture medium. The intracellular SDMANF protein level was up-regulated in response to both a Golgi/ER transport inhibitor and bacterial lipopolysaccharides (LPS). Upon LPS challenge, transfected cells revealed a decreased caspase-3 activity and increased cell viability with no inducible Bax expression compared to the wild type. These results suggest a deep evolutionary original cytoprotective role of MANF, at the crossroads of innate immune and apoptotic pathways, of which a neurotrophic function might have arisen later in metazoan evolution., Highlights • Identification of the MANF homolog SDMANF in the ancestral-like clade Porifera (sponges). • Co-localization of SDMANF in bacteriocytes of sponge tissue with a toll-like receptor. • Up-regulation of the intracellular SDMANF protein level but not BAX in LPS-challenged SDMANF-transfected human cells. • Enhanced viability, reduced caspase activity of LPS-challenged SDMANF-transfected human cells. • An evolutionary ancient cytoprotective role of SDMANF at the crossroads of innate immune and apoptotic pathways is proposed.

Marine organisms especially those that live sessile, as sponges, are well known to have specific relationships with a great variety of microorganisms including bacteria and fungi. As most simple metazoan phylum, the Porifera, which emerged first during the transition from the non-Metazoa to the Metazoa from the common ancestor, comprise wide arrays of recognition molecules, both for Gram-negative bacteria and for Gram-positive bacteria as well as for fungi. They react specifically with effector molecules to inhibit or kill the invading microorganisms. The elicitation and the subsequent effector reactions of the sponges towards these microbes are outlined. However, besides of the elimination of bacteria and fungi, some of those taxa are kept as symbionts of the sponges, allowing them, for example, to accumulate the essential element manganese or to synthesize carotinoids. The sponges produce low-molecular-weight bioactive compounds, secondary metabolites, to eliminate the microorganisms. In addition, they are armed with cationic antimicrobial peptides allowing them to defend against invasive microorganisms and, in parallel, to kill or repel also metazoan invaders. The broad range of chemically and functionally different compounds qualifies the Porifera as the most important animal phylum to be exploited as a source for the isolation of new potential drugs. First molecular biological strategies have been outlined to obtain those compounds in a sustainable way, by producing them recombinantly. © 2012 Springer Science+Business Media B.V.

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula as a model, quantitative enzymatic studies revealed that both the native and the recombinant enzyme display in vitro the same biosilica-forming activity as the enzyme involved in spicule formation in vivo. Monomeric silicatein molecules assemble into filaments via fractal intermediates, which are stabilized by the silicatein-interacting protein silintaphin-1. Besides the silicateins, a silica-degrading enzyme silicase acting as a catabolic enzyme has been identified. Growth of spicules proceeds in vivo in two directions: first, by axial growth, a process that is controlled by evagination of cell protrusions and mediated by the axial filament-associated silicateins; and second, by appositional growth, which is driven by the extraspicular silicateins, a process that provides the spicules with their final size and morphology. This radial layer-by-layer accretion is directed by organic cylinders that are formed around the growing spicule and consist of galectin and silicatein. The cellular interplay that controls the morphogenetic processes during spiculogenesis is outlined.

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the β,β-carotene-15,15′-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only β-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade β-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with β-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from β-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin.

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.

Biomineralization processes are characterized by controlled deposition of inorganic polymers/minerals mediated by functional groups linked to organic templates. One metazoan taxon, the siliceous sponges, has utilized these principles and even gained the ability to form these polymers/minerals by an enzymatic mechanism using silicateins. Silicateins are the dominant protein species present in the axial canal of the skeletal elements of the siliceous sponges, the spicules, where they form the axial filament. Silicateins also represent a major part of the organic components of the silica lamellae, which are cylindrically arranged around the axial canal. With the demosponge Suberites domuncula as a model, quantitative enzymatic studies revealed that both the native and the recombinant enzyme display in vitro the same biosilica-forming activity as the enzyme involved in spicule formation in vivo. Monomeric silicatein molecules assemble into filaments via fractal intermediates, which are stabilized by the silicatein-interacting protein silintaphin-1. Besides the silicateins, a silica-degrading enzyme silicase acting as a catabolic enzyme has been identified. Growth of spicules proceeds in vivo in two directions: first, by axial growth, a process that is controlled by evagination of cell protrusions and mediated by the axial filament-associated silicateins ; and second, by appositional growth, which is driven by the extraspicular silicateins, a process that provides the spicules with their final size and morphology. This radial layer-by-layer accretion is directed by organic cylinders that are formed around the growing spicule and consist of galectin and silicatein. The cellular interplay that controls the morphogenetic processes during spiculogenesis is outlined.

The synthesis of siliceous spicules in both demosponges and hexactinellids is enzymatically driven via silicatein. This enzyme exists both intra-spicularly and in the extra-spicular space. It catalyzes the formation of bio-silica constituting the silica lamellae that are formed during the appositional (layer-by-layer) growth of the spicules. The extent of (bio-silica forming) activity of silicatein from the demosponge Suberites domuncula measured in vitro reflects the amount of bio-silica required for the formation of spicules in vivo. It is shown that during growth and maturation of the spicules in demosponges a bio-fusion process occurs that results in an intra-spicular sintering of the silica lamellae to form compact silica rods. The morphological characterization of the globular asters (microscleres) from the demosponge Geodia cydonium revealed that these spherical spicules are formed from a tuft of silicatein filaments which radiate from a common origin. While in demosponges a complete intra-spicular bio-sintering/bio-fusion process occurs, this process is incomplete in hexactinellid spicules. There, only the most inner lamellae of the spicules fuse leaving the more peripheral silica lamellae separate. However, within this class of sponges several families (example Euplectella aspergillum) show a bio-sintering process between individual spicules. There, bio-silica is secondarily deposited onto mature spicules, giving rise to an ordered array of bio-silica bridges. Furthermore we report that for the formation of the strong and stiff bio-silica skeleton of sponges a hardening process is required that is (presumable) driven by cell-membrane bound aquaporin channels which allow the removal of water, released during the bio-silica polycondensation reaction.

In the present study we examined material from the Ashikule Basin of Tibet. Chemical analyses were performed by use of energy dispersive X-ray spectroscopy and electron probe microanalysis to clarify whether the varnish layers that had developed on the surface of the rhyolite are indeed composed of varnish bodies and silica glaze. Electron microscopic analyses revealed that the surface of the varnish is covered both by filamentous hyphae bacterial and cocci-shaped forms. Within the varnish mineral layer in those samples two forms of bacteria-like microorganisms exist; cocci as tightly packed bacterial aggregates [within varnish bodies], and bacillus-like microorganisms [within the varnish matrix, that surrounds the varnish bodies]. The bacillus-like forms are embedded in a network of filaments that have diameters between 35 and 45 nm. These bacilli with the surrounding filaments are assumed to have been involved in biofilm formation (synthesis of extracellular polymeric substances) prior to their live burial. We concluded that the formation of the varnish layers was for the most part biogenically driven by microorganisms.

Biomineralization is the process by which living organisms or organic matrices produced by them initiate and structure deposition of inorganic polymers/minerals. Deep-sea polymetallic nodules and crusts have recently been recognized as biominerals that are formed around bio-seeds; these deposits are of economic value. A detailed understanding of their formation will contribute to their sustainable exploitation in the future. Polymetallic nodules grow concentrically around discrete nuclei that have recently been described as bio-seeds formed from microorganisms, diatoms, or coccoliths. In the present study, polymetallic nodules from the Clarion-Clipperton Zone have been analyzed. It is described that the approximately 5-cm large polymetallic nodules are composed of micronodules (size of 100-450 μm) that aggregated to nests (2-3 mm). High-resolution scanning electron microscopy (HR-SEM) and high-resolution energy dispersive X-ray (HR-EDX) spectroscopic analyses revealed that the micronodules are composed of discrete layers of Mn and Fe. Imprints of microorganisms/microbe-like assemblies are found in the Mn-rich regions of the micronodules. HR-SEM/EDX analyses confirmed that these microorganisms are surrounded by a Mn-rich environment. These findings strongly suggest that those organisms acted as bio-seeds that allowed the deposition of Mn(IV) minerals which in turn helped Fe minerals to associate. Hence, these data support the concept that the growth of the polymetallic nodules starts as a biomineral and is completed by genuine mineralic depositions. It is expected that these data will contribute to the development of strategies for a sustainable exploitation of the polymetallic nodules.

In nature, mineralization of hard tissues occurs due to the synergistic effect of components present in the organic matrix of these tissues, with templating and catalytic effects. In Suberites domuncula, a well-studied example of the class of demosponges, silica formation is mediated and templated by an axial proteinaceous filament with silicatein-α, one of the main components. But so far, the effect of other organic constituents from the proteinaceous filament on the catalytic effect of silicatein-α has not been studied in detail. Here we describe the synthesis of core-shell TiO(2)@SiO(2) and TiO(2)@ZrO(2) nanofibers via grafting of silicatein-α onto a TiO(2) nanowire backbone followed by a coassembly of silintaphin-1 through its specifically interacting domains. We show for the first time a linker-free, one-step funtionalization of metal oxides with silicatein-α using glutamate tag. In the presence of silintaphin-1 silicatein-α facilitates the formation of a dense layer of SiO(2) or ZrO(2) on the TiO(2)@protein backbone template. The immobilization of silicatein-α onto TiO(2) probes was characterized by atomic force microscopy (AFM), optical light microscopy, and high-resolution transmission electron microscopy (HRTEM). The coassembly of silicatein-α and silintaphin-1 may contribute to biomimetic approaches that pursue a controlled formation of patterned biosilica-based biomaterials.

In this study we demonstrate that the demosponge Suberites domuncula harbors a Mn(II)-oxidizing bacterium, a Bacillus strain, termed BAC-SubDo-03. Our studies showed that Mn(II) stimulates bacterial growth and induces sporulation. Moreover, we show that these bacteria immobilize manganese on their cell surface. Comparison of the 16S rDNA sequence allowed the grouping of BAC-SubDo-03 to the Mn-precipitating bacteria. Analysis of the spore cell wall revealed that it contains an Mn(II)-oxidizing enzyme. Co-incubation studies of BAC-SubDo-03 with 100 mu M MnCl(2) and >1 mu M of CuCl(2) showed an increase in their Mn(II)-oxidizing capacity. In order to prove that a multicopper oxidase-like enzyme(s) (MCO) exists in the cell wall of the S. domuncula-associated BAC-SubDo-03 Bacillus strain, the gene encoding this enzyme was cloned (mnxG-SubDo-03). Sequence alignment of the deduced MCO protein (MnxG-SubDo-03) revealed that the sponge bacterium clusters together with known Mn(II)-oxidizing bacteria. The expression of the mnxG-SubDo-03 gene is under strong control of extracellular Mn(II). Based on these findings, we assume that BAC-SubDo-03 might serve as a Mn reserve in the sponge providing the animal with the capacity to detoxify Mn in the environment. Applying the in vitro primmorph cell culture system we could demonstrate that sponge cells, that were co-incubated with BAC-SubDo-03 in the presence of Mn(II), show an increased proliferation potential.

The skeleton of siliceous sponges (phylum Porifera: classes Demospongiae and Hexactinellida), composed of tightly interacting spicules that assemble to a genetically fixed scaffold, is formed of bio-silica. This inorganic framework with the quality of quartz glass has been shown to operate as light waveguide in vitro and very likely has a similar function in vivo. Furthermore, the molecular toolkit for endogenous light generation (luciferase) and light/photon harvesting (cryptochrome) has been identified in the demosponge Suberites domuncula. These three components of a light signaling system, spicules—luciferase—cryptochrome, are concentrated in the surface layers (cortex) of the poriferan body. Specimens from which this cortex has been removed/ablated do not emit light. However, with regeneration and reconstitution of the cortex the animals re-gain the capacity to flash light. This newly discovered characteristic of sponges to generate light prompted us to investigate the genetic basis for the endogenous light signaling system. As a potential transcription factor involved in the expression of luciferase and cryptochrome, a SOX-related protein has been identified. In dark-adapted animals or in tissue from below the cortex region, the medulla, no gene or protein expression of SOX-related protein, luciferase, and cryptochrome could be detected. However, during the regeneration of the cortex, a stage-specific expression pattern was recorded: SOX-related protein > luciferase > cryptochrome. We conclude that a flashing light signaling circuit exists, which might control the retinoic acid-induced differentiation of stem cells into pulsating and contracting sponge cells, that is, pinacocytes and myocytes. J. Cell. Biochem. 111: 1377–1389, 2010. © 2010 Wiley-Liss, Inc.

The sponge protein silicatein is the first enzyme that has been described to form an inorganic polymer (silica) from a monomeric precursor (tetraethoxysilane or orthosilicic acid). The models proposed for silicatein-mediated silica formation are mainly based on the use of synthetic substrates (hydrolytic cleavage of tetraethoxysilane to silanol compounds) or only consider the formation of less reactive silicic acid dimers (disilicic acid). Here we propose a new model for the catalytic mechanism of silicatein that leads to the formation of reactive, cyclic silicic acid species (trisiloxane rings and higher-membered siloxane rings) which easily promote the silica polycondensation reaction.

Earlier studies have demonstrated that biosilica, synthesized by the enzyme silicatein, induces hydroxyapatite formation in osteoblast-like SaOS-2 cells. Here we study the effect of biosilica on the expressions of osteoprotegerin [OPG] and the receptor activator for NF-κB ligand [RANKL] in the SaOS-2 cell model. We show that during growth of SaOS-2 cells on biosiliceous matrices hydroxyapatite formation is induced, while syntheses of cartilaginous proteoglycans and sulfated glycosaminoglycans are down-regulated. Furthermore, quantitative real-time RT-PCR analysis revealed a strong time-depended increase in expression of OPG in biosilica exposed SaOS-2 cells while the steady-state expression level of RANKL remained unchanged. These results have been corroborated on the protein level by ELISA assays. Therefore, we propose that biosilica stimulated OPG synthesis in osteoblast-like cells counteracts those pathways that control RANKL expression and function (e.g. maturation of pre-osteoclasts and activation of osteoclasts). Hence, the data obtained in the present study reveal the considerable biomedical potential of biosilica for treatment and prophylaxis of osteoporotic disorders.

Most forms of multicellular life have developed a calcium-based skeleton, while only a few specialized organisms complement their body plan with silica, such as sponges (phylum Porifera). However, the way in which sponges synthesize their silica is exceptional. They use an enzyme, silicatein, for the polymerization/polycondensation of silica, and thereby form their highly resistant and stabile massive siliceous skeletal elements (spicules). During this biomineralization process (i.e., biosilicification), hydrated amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. This peculiar phenomenon has been comprehensively studied in recent years and by several approaches; the molecular background was explored to create tools that might be employed for novel bio-inspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it gains its final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics.

Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula shares considerable similarities with its metazoan homologs, ranging from conserved exon/intron structure to presence of protein-interaction domains. Whereas sponge tissue shows a low steady-state level, SURVL expression was significantly upregulated in rapidly proliferating primmorph cells. In addition, challenge of tissue and primmorphs with heavy metal or lipopeptide stimulated SURVL expression, concurrent with the expression of a newly discovered caspase. Complementary functional analyses in transfected HEK-293 cells revealed that heterologous expression of a SURVL-EFGP fusion not only promotes proliferation but also enhances resistance to cadmium-induced cell death. Taken together, these results suggest both a deep evolutionary conserved dual role of survivin and an equally conserved central position in the interconnected pathways of cell cycle and apoptosis.

The gold standard for bone reconstruction is the use of autogeneic grafts from various donor regions, since they possess osteoinductive as well as osteoconductive potential. Only a few synthetic materials possess/display properties that allow optimal bone reconstitution. Previously, we showed that the natural product, bio-silica, comprises osteoinductive, and probably also osteoconductive activity. Bio-silica is formed enzymatically via silicatein; this enzyme has been isolated from siliceous sponges and has also been cloned and prepared recombinantly. In the present study, silicatein was encapsulated together with its substrate, sodium metasilicate, in poly(D,L-lactide)/poly(vinyl pyrrolidone)-based microspheres, termed silicatein-and-silica-containing microspheres (SSMs). The deposition of silica and the kinetics of silicatein release from the microspheres are given. Furthermore, SSMs were successfully embedded in a poly(vinyl pyrrolidone)/starch-based matrix, termed plastic-like filler matrix containing silicic acid (PMSA). A blend of SSM and PMSA forms a biocompatible, moldable, and biodegradable functional implant material that hardens at a controlled and clinically suitable rate within approximately 30 min to 6 h to implants that were tightly integrated in artificial defects of rabbit femurs. Until now no toxic reactions caused by the silicatein have been observed in vitro or in vivo. We assume that the data given here contribute to a successful introduction of the silicatein/bio-silica-based implant materials to the field of regenerative medicine.

Based on the light-reactive behavior of siliceous sponges, their intriguing quartz glass-based spicular system and the existence of a light-generating luciferase [Muller WEG et al. (2009) Cell Mol Life Sci 66, 537–552], a protein potentially involved in light reception has been identified, cloned and recombinantly expressed from the demosponge Suberites domuncula. Its sequence displays two domains characteristic of cryptochrome, the N-terminal photolyase-related region and the C-terminal FAD-binding domain. The expression level of S. domuncula cryptochrome depends on animal’s exposure to light and is highest in tissue regions rich in siliceous spicules; in the dark, no cryptochrome transcripts/translational products are seen. From the experimental data, it is proposed that sponges might employ a luciferase-like protein, the spicular system and a cryptochrome as the light source, optical waveguide and photosensor, respectively.

Terra Nova, 22, 1–11, 2010 Abstract The emergence of the Metazoa can be dated back to the Neoproterozoic Era which comprises the Cryogenian Period during which two major glaciations occurred, the Sturtian and the Varanger-Marinoan. At that time, the phylum Porifera (sponges) evolved as the first animals and developed a hard skeleton. The two classes of siliceous sponges, the Hexactinellida and the Demospongiae, are already provided with the major genetic repertoire and gene regulatory networks that also exist in modern multicellular animals. Besides these metazoan innovations, the siliceous sponges display one autapomorphic character, silicatein, an enzyme which mediates bio-silica formation. Well preserved siliceous sponge fossils have been excavated from the Cambrian Burgess Shale- and Chengjiang deposits. It is concluded that it was the hard skeleton of the siliceous sponges that contributed to the successful evolution and survival of the Porifera during the last 500 Ma.

Recently two approaches were reported that addressed a vitally important problem in regenerative medicine, i. e. the successful treatment of wounds even under diabetic conditions. Accordingly, these studies with diabetic rabbits [Sarojini et al. PLoS One 2017, 12(4):e0174899] and diabetic mice [Müller et al. Polymers 2017, 9, 300] identified a novel (potential) target for the acceleration of wound healing in diabetes. Both studies propose a raise of the intracellular metabolic energy status via exogenous administration either of ATP, encapsulated into lipid vesicles, or of polyphosphate (polyP) micro-/nanoparticles. Recently this physiological polymer, polyP, was found to release metabolic energy in form of ATP into both the extra- and also intra-cellular space. In the present work the uptake mechanism of the amorphous polyP microparticles “Ca-polyP-MP” has been described and found to be a clathrin-dependent endocytosis import, based on inhibition studies with the inhibitor trifluoperazine, which blocks the clathrin-dependent endocytosis import. The experiments had been performed with SaOS-2 cells, by studying the uptake and distribution of the electron-dense particles into the cells, and with HUVEC cells, for analysis of the intracellular accumulation of polyP, visualized by fluorescent staining of polyP. Concurrently with the uptake of particular polyP the intracellular ATP level increased as well. In contrast to “Ca-polyP-MP” the soluble polyP, administered as “Na-polyP[Ca2+]”, did not cause an increase in the intracellular Ca2+ level, suggesting a different mode of action of these two forms of polyP. Based on existing data on the effect of polyP and ATP on the induction of vascularization during wound repair, both groups (Sarojini et al. and Müller et al.) propose that the acceleration of wound repair is based on an increased metabolic energy supply directly to the regenerating wound area.

Here, design of the first pathogen-mimicking metal oxide nanoparticles with the ability to enter cancer cells and to selectively target and activate the TLR9 pathway, and with optical and MR imaging capabilities, is reported. The immobilization of ssDNA (CpG ODN 2006) on MnO nanoparticles is performed via the phosphoramidite route using a multifunctional polymer. The multifunctional polymer used for the nanoparticle surface modification not only affords a protective organic biocompatible shell but also provides an efficient and convenient means for loading immunostimulatory oligonucleotides. Since fluorescent molecules are amenable to photodetection, a chromophore (Rhodamine) is introduced into the polymer chain to trace the nanoparticles in Caki-1 (human kidney cancer) cells. The ssDNA coupled nanoparticles are used to target Toll-like receptors 9 (TLR9) receptors inside the cells and to activate the classical TLR cascade. The presence of TLR9 is demonstrated independently in the Caki-1 cell line by western blotting and immunostaining techniques. The magnetic properties of the MnO core make functionalized MnO nanoparticles potential diagnostic agents for magnetic resonance imaging (MRI) thereby enabling multimodal detection by a combination of MR and optical imaging methods. The trimodal nanoparticles allow the imaging of cellular trafficking by different means and simultaneously are an effective drug carrier system.

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.

Polymetallic/ferromanganese nodules (Mn-nodules) provide a rich source for manganese. It is not yet known if the nodules have a biogenic or an abiogenic origin. Here we applied the technique of high-resolution scanning electron microscopy, in combination with energy dispersive X-ray spectroscopical (EDX) analysis, to trace the existence of microbial biofilms. Two spatially separated assemblies exist, the exolithobiontic- and endolithobiontic colonizations. The exolithobiontic colonization is seen in the micro-canals, which traverse the outer surface layer of the nodules and are formed by elongated filamentous organisms, which show no signs of mineralization. In the center of the nodules three types of endolithobiontic microbial biofilms exist: first, cone-like microorganisms forming biofilms, second stone/pillar-like microorganisms and finally paving stone-like, hexagonal microorganisms. All are covered by brick-like mineral deposits. By EDX analysis we could measured the highest relative level of carbon (C) with respect to manganese (Mn) and sodium on the microorganisms. Our data are in perfect agreement with the assumption that the Mn deposits in the nodules are of biogenic origin. In a first approach, DNA from microorganisms from the interior of those Mn-nodules was isolated by PCR, and sequenced with respect to the 16S ribosomal RNA gene. Sequence comparison revealed that the sequence from the Mn-nodule, studied here, shares highest similarity to a bacterium living in soil, rich in iron and manganese. We propose that the microorganisms form a biofilm within the nodules onto which Mn is deposited due to an oxidation from Mn(II) to Mn(III)/Mn(IV).

Polymetallic/ferromanganese nodules (Mn-nodules) have been assigned a huge economic potential since they contain considerable concentrations of manganese, copper, nickel, iron, and cobalt. It has been assumed that they are formed by, besides hydrogenous, nonbiogenic processes, biogenic processes based on metabolic processes driven by microorganisms. In the present study, we applied the techniques of digital optical microscopy and high-resolution scanning electron microscopy to search for microorganisms in Mn-nodules. They were collected from the Clarion-Clipperton Zone in the Eastern Pacific Ocean and are composed of Mn (23.9%), Cu (0.69%), Ni (1.02%), Fe (10.9%), and Co (0.29%). These Mn-nodules, between 2.3 and 4.8 cm, show a distinct lamination; they are composed of small-sized micronodules, 100 to 450 microm in size, which are bound together by an interstitial whitish material. In the micronodules, a dense accumulation of microorganisms/bacteria could be visualized. Only two morphotypes exist: (1) round-shaped cocci and (2) elongated rods. The cocci (diameter: approximately 3.5 microm) are arranged in bead-like chains, while the rods (approximately 2 x 0.4 microm) are arranged either as palisades or in a linear row. Energy-dispersive X-ray spectroscopy analyses showed that the areas rich in microorganisms/bacteria are also rich in Mn, while in regions where no microorganisms are found, the element Si is dominant. We suggest that growth of the Mn-nodules starts with the formation of "micronodules." The formation of micronodules is assumed to be mediated by microorganisms. After accretion of biogenic and additional nonbiogenic minerals, the micronodules assemble to large nodules on the sea floor through additional inclusion of nonbiogenic material.

Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2'-5'-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in a crude extract from L. baicalensis by application of a novel separation procedure based on polymer coated ferromagnetic nanoparticles. The particles were derivatized with a synthetic double-stranded RNA [dsRNA], synthetic poly(I:C), a known allosteric activator of the latent (2-5)A synthetase. These particles were used to separate a single 35kDa protein from a crude extract of L. baicalensis, which cross-reacted with antibodies raised against the sponge enzyme. In situ hybridization studies revealed that highest expression of the gene is seen in cells surrounding the aquiferous canals. Finally primmorphs, an in vitro cell culture system, from L. baicalensis were exposed to poly(I:C); they responded to this dsRNA with an increased expression of the (2-5)A synthetase gene already after a 1-day incubation period. We conclude that sponges contain the (2-5)A synthetase antiviral protection system.

Some sponges [phylum Porifera], e.g. the demosponges Lubomirskia baicalensis or Axinella polypoides, show an arborescent growth form. In the freshwater sponge L. baicalensis this morphotype is seen mostly in depths below 4 m while in more shallow regions it grows as a crust. The different growth forms are determined in nature very likely by water current and/or light. The branches of this species are composed of modules, arranged along the apical-basal axis. The modules are delimited by a precise architecture of the spicule bundles; longitudinal bundles originate from the apex of the earlier module, while at the basis of each module these bundles are cross-linked by traverse bundles under formation of annuli. Genes encoding putative morphogenetic factors, myotrophin and epidermal growth factor (EGF)-like molecules, and one gene of an antagonist for the Wnt signaling pathway, the soluble frizzled molecule, have been identified and characterized. Their expression levels as well as those of silicatein, one major spicule-forming molecule, have been studied in the crusts and the modules. The data revealed that at the apices of each module higher level of expression of myotrophin and EGF can be detected, while the base of each module is characterized by a high steady-state expression level of soluble frizzled molecule. These results suggest that module formation in L. baicalensis is controlled by a tuned interaction of agonistic (e.g., myotrophin and EGF) as well as antagonistic morphogenetic factors (e.g., soluble frizzled molecule).

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula ; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.

Inspired by the intermolecular cross-linking of mussel foot proteins and their adhesive properties, tyrosinase has been used to modify recombinant silicatein. DOPA/DOPAquinone-mediated cross-linking and interfacial interactions enhanced both self-assembly of silicatein building blocks and templating of core–shell silica spheres, resulting in fluorescent biomimetic silicatein–silica hybrid mesofibers.

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for Gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates Gram-negative bacteria; it is inactive against Gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against Gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.

SUMMARY In Demospongiae (phylum Porifera) the formation of the siliceous skeleton,composed of spicules, is an energetically expensive reaction. The present study demonstrates that primmorphs from the demosponge Suberites domuncula express the gene for arginine kinase after exposure to exogenous silicic acid. The deduced sponge arginine kinase sequence displays the two characteristic domains of the ATP:guanido phosphotransferases; it can be grouped to the `usual' mono-domain 40 kDa guanidino kinases (arginine kinases). Phylogenetic studies indicate that the metazoan guanidino kinases evolved from this ancestral sponge enzyme; among them are also the `unusual'two-domain 80 kDa guanidino kinases. The high expression level of the arginine kinase gene was already measurable 1 day after addition of silicic acid by northern blot, as well as by in situ hybridization analysis. Parallel determinations of enzyme activity confirmed that high levels of arginine kinase are present in primmorphs that had been exposed for 1-5 days to silicic acid. Finally, transmission electron-microscopical studies showed that primmorphs containing high levels of arginine kinase also produce siliceous spicules. These data highlight that silicic acid is an inorganic morphogenetic factor that induces the expression of the arginine kinase, which in turn probably catalyzes the reversible transfer of high-energy phosphoryl groups.

In a preceding study it has been reported that the freshwater sponge Lubomirskia baicalensis, living in Lake Baikal (East Siberia), is composed of spicules forming a characteristic pattern which follows radiate accretive growth. Here we report that the spicules are synthesized by the enzyme silicatein, a protein which is related to cathepsin L. The cDNAs for silicatein and the related cathepsin L were isolated and used as probes to show that the mRNA levels of silicatein in the bases of the spicule skeleton of the animals are low, while the mRNA level of cathepsin L in this region exceeds that of the growing zone. This is the first comprehensive study on the importance of the axial filament/silicatein as an essential structural and functional component determining the growth and stability of demosponge spicules.

Silicon is, besides oxygen, the most abundant element on earth. Only two taxa use this element as a major constituent of their skeleton, namely sponges (phylum Porifera) and unicellular diatoms. Results from combined cytobiological and molecular-biological techniques suggest that, in the demosponge Suberites domuncula, silicic acid is taken up by a transporter. Incubation of cells with the fluorescent silica tracer PDMPO [2-(4-pyridyl)-5-{; ; ; [4-(2-dimethylaminoethylaminocarbamoyl)methoxy]phenyl}; ; ; -oxazole] showed a response to silicic acid by an increase in fluorescence ; this process is temperature-dependent and can be blocked by DIDS (4, 4-di-isothiocyanatostilbene-2, 2-disulphonic acid). The putative NBC (Na+/HCO3-) transporter was identified, cloned and analysed. The deduced protein comprises all signatures characteristic of those molecules, and phylogenetic analysis also classifies it to the NBC transporter family. This cDNA was used to demonstrate that the expression of the gene is strongly up-regulated after treatment of cells with silicic acid. In situ hybridization demonstrated that the expression of the sponge transporter occurs in those cells that are located adjacent to the spicules (the skeletal element of the animal) or in areas in which spicule formation occurs. We conclude that this transporter is involved in silica uptake and have therefore termed it the NBCSA {; ; ; Na+/HCO3-[Si(OH)(4)]}; ; ; co-transporter

The progress in molecular and cell biology has enabled a rational exploitation of the natural resources of the secondary metabolites and biomaterials from sponges (phylum Porifera). It could be established that these natural substances are superior for biomedical application to those obtained by the traditional combinatorial chemical approach. It is now established that the basic structural and functional elements are highly conserved from sponges to the crown taxa within the Protostomia (Drosophila melanogasterandCaenorhabditis elegans) and Deuterostomia (human); therefore, it is obvious that the molecular etiology of diseases within the metazoan animals have a common basis. Hence, the major challenge for scientists studying natural product chemistry is to elucidate the target(s) of a given secondary metabolite, which isper sehighly active and selective. After this step, the potential clinical application can be approached. The potential value of some selected secondary metabolites, all obtained from sponges and their associated microorganisms, is highlighted. Examples of compounds that are already in medical use (inhibition of tumor/virus growth [arabinofuranosyl cytosine and arabinofuranosyl adenine]), or are being considered as lead structures (acting as cytostatic and anti-inflammatory secondary metabolites [avarol/avarone], causing induction of apoptosis [sorbicillactone]) or as prototypes for the interference with metabolic pathways common in organisms ranging from sponges to humans (modulation of pathways activated by fungal components [aeroplysinin], inhibition of angiogenesis [2-methylthio-1,4-napthoquinone], immune modulating activity [FK506]) are discussed in this study. In addition, bioactive proteins from sponges are listed (antibacterial activity [pore-forming protein and tachylectin]). Finally, it is outlined that the skeletal elements—the spicules—serve as blueprints for new biomaterials, especially those based on biosilica, which might be applied in biomedicine. These compounds and biomaterials have been isolated/studied by members of the German Center of ExcellenceBIOTECmarin. The goal for the future is to successfully introduce some of these compounds in the treatment of human diseases in order to raise the public awareness on the richness and diversity of natural products, which should be sustainably exploited for human benefit.

It is now well established that all metazoan phyla derived from one common ancestor, the hypothetical Urmetazoa. Due to the basal position of Porifera (Demospongiae) in the phylogenetic tree of Metazoa, studies on the mechanisms controlling the development of these animals can provide clues on the understanding of the origin of multicellular animals and on how the first organization of the body plan evolved. In this report we describe the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula. The phylogenetic analysis classifies one into the subfamily of Brachyury, Sd-Bra, and the second into the Tbx2 subfamily, Sd-Tbx2. Analyses of the Sd-Bra and Sd-Tbx2 sequences and their intron-exon structures demonstrate their basal position in the phylogeny of the T-box family, and allows us to hypothesize a model of the phylogenetic evolution of all T-box genes. Furthermore, we report the presence of two different products of alternative splicing of Sd-Bra, and demonstrate that they exist in different phosphorylation and glycosylation states in the sponge tissue. Sd-Bra expression in tissue and 3D-cell aggregates (primmorphs) is analyzed, suggesting that Sd-Bra might also have a role in Porifera morphogenesis.

Molecular data on development/differentiation and on comparative genomics allow insights into the genetic basis of the evolution of a bodyplan. Sponges (phylum Porifera) are animals that are the (still extant) stem group with the hypothetical Urmetazoa as the earliest common ancestor of all metazoans; they possess the basic features of the characteristic metazoan bodyplan also valid for the animals of the crown taxa. Here we describe three homeobox genes from the demosponge Suberites domuncula whose deduced proteins (HOXa1_SUBDO, HOXb1_SUBDO, HOXc1_SUBDO) are to be grouped with the Antennapedia class of homeoproteins (subclasses TIx-Hox11 and NK-2). In addition, a cDNA encoding a LIM/homeobox protein has been isolated which comprises high sequence similarity to the related LIM homeodomain (HD) proteins in its LIM as well as in its HD domains. To elucidate the potential function of these proteins in the sponge a new in vitro system was developed. Primmorphs which are formed from dissociated cells were grown on a homologous galectin matrix. This galectin cDNA was cloned and the recombinant protein was used for the preparation of the matrix. The galectin/polylysine matrix induced in primmorphs the formation of channels, one major morphogenetic process in sponges. Under such conditions the expression of the gene encoding the LIM/homeobox protein is strongly upregulated, while the expression of the other homeobox genes remains unchanged or is even downregulated. Competition experiments with galactosylceramides isolated from S. domuncula were performed. They revealed that a β-galactosylceramide, named Sdgal-1, prevented the expression of the LIM gene on the galectin matrix, while Sdgal-2, a diglycosylceramide having a terminal α-glycosidically linked galactose, caused no effect on the formation of channels in primmorphs or on LIM expression. This study demonstrates for the first time that an extracellular matrix molecule, galectin, induces a morphogenetic process in sponges which is very likely caused by a LIM/homeobox protein. Furthermore, a new model is introduced (galectin-caused channel formation in sponge primmorphs) to investigate basic pathways, thus allowing new insights into the functional molecular evolution of Metazoa.

Sponges are sessile filter feeders that are exposed to toxic compounds/xenobiotics and a huge number of diverse pro- and eukaryotic organisms occurring in the aqueous milieu. To cope with these threats, sponges have developed a variety of chemical and immunological defense systems. Among them are potent secondary metabolites or toxins. The demosponge Suberites domuncula contains the bioactive compound okadaic acid; concentrations of ≈50 ng/g (≈60 nM) of wet tissue have been quantified by LC/MS. In the present study, we sought to determine whether okadaic acid is beneficial for this sponge. Antibodies were raised against okadaic acid and used to localize this compound in situ. It could be demonstrated that the polyclonal antibodies/antiserum stained the sponge cells (endopinacocytes) which form the epithelium of the lacunae and the water channels. They also reacted with bacteria present in sponge tissue. Okadaic acid was found to augment the immune response of S. domuncula against the bacterial endotoxin lipopolysaccharide (LPS). If primmorphs, a special form of 3-D aggregates, are incubated with LPS at a concentration of 3 µg/ml, a reduction in the incorporation rate of [3H] phenylalanine is measured. In the presence of okadaic acid, this effect is measured already at a concentration of 1 µg/ml, suggesting that okadaic acid sensitizes this (immune) response. Okadaic acid is a known inhibitor of protein phosphatases which, as shown here, cause an increased phosphorylation of p38, a central kinase of the MAP kinase pathway; this may explain the sensitive response of the sponge cells to LPS. At higher concentrations, okadaic acid induces apoptosis, as was demonstrated here by a differential expression of the Bcl-2 homolog and the caspase gene. It is concluded that okadaic acid has two functions in the sponge; first, at low concentrations (

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related proteins were isolated; they comprise the characteristic structure known from caspases of other metazoan phyla. The two cDNAs are assumed to originate from one gene by alternative splicing; the longer form comprises a caspase recruitment domain (CARD), whereas the shorter one is missing CARD. The expression of sponge caspase genes is up-regulated during allograft rejection. In vivo incubation experiments with Ac-DEVD-CHO (a caspase-3 inhibitor) showed a reduction of apoptotic DNA fragmentation, whereas Ac-LEHD-CHO (an inhibitor of caspase-9) caused no effect. It is concluded, that for the establishment of the metazoan body plan, both the adhesion molecules and the apoptotic molecules (described here) were essential prerequisites.