New putative phenol oxidase in ascidian blood cells.

The phenol oxidase system is ancient and ubiquitously distributed in all living organisms. In various groups it serves for the biosynthesis of pigments and neurotransmitters (dopamine), defence reactions and tissue hardening. Ascidians belong to subphylum Tunicata, which is considered the closest living relative to Vertebrates. Two phenol oxidases previously described for ascidians are vertebrate-like and arthropod-like phenol oxidases. In our present study, we described a new ascidian protein, Tuphoxin, with putative phenol oxidase function, which bears no sequence similarity with two enzymes described previously. The closest related proteins to Tuphoxin are mollusc haemocyanins. Unlike haemocyanins, which are oxygen transporting plasma proteins, Tuphoxin is synthesised in ascidian blood cells and secreted in the extracellular matrix of the tunic-ascidian outer coverings. Single mature transcript coding for this phenol oxidase can give several protein products of different sizes. Thus limited proteolysis of the initial protein is suggested. A unique feature of Tuphoxins and their homologues among Tunicata is the presence of thrombospondin first type repeats (TSP1) domain in their sequence which is supposed to provide interaction with extracellular matrix. The finding of TSP1 in the structure of phenol oxidases is new and we consider this to be an innovation of Tunicata evolutionary lineage.

[Culture of mussel Mytiuls edulis I. mantle cells].

To date, cell lines derived from marine invertebrates have not been available. Hence primary cell cultures serve as model systems for various experiments. In present study we established primary culture of mussel Mytilus edulis L. mantle cells. Cells were isolated by means of explant culture or enzymatic dissociation of mantle tissue. They maintained viability up to 22 months regardless of culture initiation method. In course of culturing, cells, which were transferred onto new plates, successfully attached to a new surface. Physiological activity of cultured cells was also confirmed by formation of crystals, which appeared after 4-6 months. After continuous time of culturing, mantle cells can be cryopreserved using 5 % DMSO with post-freezing survival up to 50%. These results demonstrate that M. edulis mantle cells can maintain viability and physiological activity for exceptionally long time and can be cryopreserved for further examination.