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.

Hsp70 in the atrial neuroendocrine units of the snail, Achatina fulica.

Heat shock proteins (Hsps) are evolutionary conserved peptides well known as molecular chaperones and stress proteins. Elevated levels of extracellular Hsps in blood plasma have been observed during the stress responses and some diseases. Information on the cellular sources of extracellular Hsps and mechanisms regulating their release is still scanty. Here we showed the presence and localization of Hsp70 in the neuroendocrine system in the atrium of the snail, Achatina fulica. The occurrence of the peptide in snail atrium lysate was detected by Western blot analysis. Immunoperoxidase and immunogold staining demonstrated that Hsp70-immunoreactivity is mainly confined to the peculiar atrial neuroendocrine units which are formed by nerve fibers tightly contacted with large granular cells. Immunolabelling intensity differed in morphologically distinct types of secretory granules in the granular cells. The pictures of exocytosis of Hsp70-immunolabeled granules from the granular cells were observed. In nerve bundles, axon profiles with Hsp70-immunoreactive and those with non-immunoreactive neurosecretory granules were found. In addition, Hsp70-like material was also revealed in the granules of glia-interstitial cells that accompanied nerve fibers. Our findings provide an immuno-morphological basis for a role of Hsp70 in the functioning of the neuroendocrine system in the snail heart, and show that the atrial granular cells are a probable source of extracellular Hsp70 in the snail hemolymph.