Dinoflagellates from marine algal blooms produce neurotoxic compounds: effects on free calcium levels in neuronal cells and synaptosomes.

In this report, evidence is presented that the marine unicellular eukaryotic dinoflagellates can cause neurotoxicity very likely by an increase in intracellular free calcium ions ([Ca(2+)](i)). Determinations of the effects of culture supernatants from different clones of the dinoflagellate Alexandrium sp. isolated from algal blooms on the viability of rat primary neuronal cells revealed that all clones tested were toxic for these cells. In addition, all Alexandrium clones tested, except for A. ostenfeldii BAH ME-141, were found to be toxic for rat pheochromocytoma PC12 cells. No toxicity was observed for culture supernatants from Gonyaulax and Coolia monotis. Calcium ions are important in the process of apoptotic cell death; our studies revealed that the dinoflagellate supernatants from A. lusitanicum K2, A. lusitanicum BAH ME-091, and A. tamarense 1M caused an increase in [Ca(2+)](i) levels in both PC12 cells and primary neuronal cells. These dinoflagellate supernatants, as well as the A. tamarense ccmp 115 supernatant, were found to cause also an increase in free calcium concentration in isolated synaptosomes. Our results suggest that the neurotoxic effects of certain dinoflagellate supernatants may be associated with disturbances in [Ca(2+)](i) levels.

Induction of heat-shock (stress) protein gene expression by selected natural and anthropogenic disturbances in the octocoral Dendronephthya klunzingeri.

Previously it was found that the expression of selected heat-shock proteins is upregulated in corals after exposure to elevated temperature. We published that HSPs are suitable markers in sponges to monitor the degree of environmental stress on these animals. In the present study the heat-shock proteins (HSPs) with a molecular weight of 90 kDa have been selected to prove their potential usefulness as biomarkers under controlled laboratory conditions and in the field. The studies have been performed with the octocoral Dendronephthya klunzingeri4.5-fold higher steady-state level of the respective mRNA. Also animals taken from stressed locations in the field showed an increased expression. The amount of HSP90 protein in D. klunzingeri was found to be strongly increased under thermal stress, or exposure to polychlorinated biphenyl (congener 118), but not after treatment with cadmium. Field studies revealed that samples taken from a nonstressed area have a low level of HSP90, but those collected from locations at which the corals are under physical stress (sedimentation through landfilling) show a high expression of HSP90. It is concluded that the chaperone HSP90 might become a suitable biomarker to monitor environmental stress on corals.

Initiation of an Aquaculture of Sponges for the Sustainable Production of Bioactive Metabolites in Open Systems: Example, Geodia cydonium.

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state.

Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship.

: The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation. From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth. The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin. Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity. The complementary DNA encoding the putative prolidase was cloned from a library of the same animal. Two different forms of cDNAs, termed SDPEPD1 and SDPEPD2, were identified, coding for the putative polypeptides PEPD_SD-1 with a molecular mass of 55,805 Da and PEPD_SD-2 with 51,684. Evidence is presented suggesting that the two different transcripts originate from the same gene but are formed by an alternative splicing event. We conclude that demosponges contain the activity as well as the gene for prolidase, a major enzyme involved in collagen metabolism, spicule formation, and cell motility. Phylogenetic analysis revealed that the sponge prolidase branches off first from the common ancestor of metazoan prolidases and later than the yeast prolidase; only distantly related are the bacterial enzymes.