Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

A new gene family of single fibrinogen domain lectins in Mytilus.

In molluscs haemolymph lectins bearing fibrinogen-like domain (FREP) act as immune pattern-recognition receptors. A full-length cDNAs of MytFREP1 and MytFREP2 cloned from haemocytes of blue mussel Mytilus edulis encoded putative polypeptides of 230 and 241 amino acids. Both polypeptides consist of signal peptide and C-terminal fibrinogen-like domain. Immune functions of these molecules may be extrapolated from the close-related and functionally characterized lectin AiFREP from bay scallop, Argopecten irradians. However, immune challenge experiments with zymosan particles, Escherichia coli bacterium and cercariae of Himasthla elongata (Trematoda) failed to modulate MytFREP1 and MytFREP2 mRNA expression in M. edulis haemocytes. Hypothetically, it argues into rather high specificity of mechanisms triggering a differential expression of MytFREP genes. The search in the EST database revealed orthologous copies for described genes and portion of relatively similar genes from two close-related mytilids, Mytilus galloprovincialis and Mytilus californianus. We document the new multigene family of FREPs from bivalves of genus Mytilus. MytFREP family currently represented by 2 genes from M. edulis, 4 genes from M. californianus and 7 genes from M. galloprovincialis.

In search of the origin of FREPs: characterization of Aplysia californica fibrinogen-related proteins.

All haemolymph lectins with uniquely juxtaposed N-terminal domain similar to the immunoglobulin superfamily (IgSF) and C-terminal fibrinogen (FBG) termed FBG-related proteins (FREP) are documented till now only in the pulmonate mollusc Biomphalaria glabrata. Using genomic WGS database we have found two FREP genes from marine opistobranch Aplysia californica named AcFREP1 and AcFREP2. The AcFREP1 and AcFREP2 mRNA molecules have been subsequently isolated from cDNA of sea hare larvae as well as adult mollusc tissues. These genes encode proteins (504 and 510aa respectively) with domain architecture typical for FREPs with two N-terminal IgSF domains and C-terminal FBG domain. Although cDNA sequences of AcFREP1 and AcFREP2 are 81% identical, their genomic structure is entirely different: AcFREP1 is intronless and AcFREP2 is encoded in four exons. These genes are paralogous pair in which AcFREP2 is a parental gene and AcFREP1 is the new transposed copy that has lost the introns (retrogene). Using RT-PCR analysis, expression of AcFREP1 and AcFREP2 was shown to be developmentally and tissue-specific and no constitutive expression in haemocytes was found. The overall frequency of nucleotide substitutions in genomic DNA trace sequences of coding region of the AcFREP1 and AcFREP2 is not higher than in the sequences of control conserved genes (actin, FMRFamide). Thus, previously reported high diversification of Biomphalaria FREP gene, BgFREP3, is not detected in Aplysia FREPs. A search for FREP homologs in other available complete genome of mollusc, Lottia gigantea (Patellogastropoda), a representative of the evolutionary earliest gastropod clade, did not reveal any DNA sequences coding for similar lectins. We suggest that unique domain architecture of FREPs is an evolutionary novelty that appeared and evolved only within one branch of Protostomata species, exclusively in heterobranch molluscs (Pulmonata and Opistobranchia).

Himasthla elongata: effect of infection on expression of the LUSTR-like receptor mRNA in common periwinkle haemocytes.

The first mollusc mRNA coding G-protein-coupled transmembrane receptor (GPcapital ES, CyrillicR), homologous to human receptors LUSTR 1 (GPR107) and LUSTR 2 (GPR108), was isolated from haemocytes of common periwinkle Littorina littorea. The analyses showed that the full-length cDNA is 1935 bp long and is predicted to encode a 614 amino acid protein (named Lit-LUSTR) with a calculated molecular mass of 69.6 kDa and theoretical isoelectric point 7.59. Pair-wise comparisons between Lit-LUSTR and LUSTR proteins from human or mouse have approximately 38% identity and 56% similarity. Lit-LUSTR clusters with LUSTR-A sub-family proteins and is a first characterization of proteins containing Lung7TM-R domain in Mollusca. Significant differences were found between the Lit-LUSTR mRNA levels in haemocytes of healthy periwinkles and those naturally infected with the echinostome trematode Himasthla elongata. Down regulated expression of the LUSTR-like receptor caused by infection illustrates modification of the haemocyte receptor system and may be attributed to the previously demonstrated greater numbers of "immature" haemocytes in the circulation of infected snails.

[The presence of nitric oxide synthase in mucosal epithelial cells of the frog urinary bladder].

In experiments on frog Rana temporaria L. urinary bladder, we investigated localization of NO-synthase (NOS) in urinary bladder slices and measured NOS activity in the suspension of mucosal epithelial cells. Intensive NADPH-diaphorase staining which is widely used as an indicator of NOS activity was found in mucosal epithelium. Almost all mucosal epithelial cells isolated in Ca2+ -free conditions demonstrated positive NADPH-diaphorase reactivity. Direct measurement of NOS activity in suspension of mucosal cells determined by the rate of conversion of L-arginine to L-citrullin showed that the enzyme activity was reduced in absence of external Ca2+ and was inhibited by L-NAME: non-specific NOS inhibitor, and 1400 W: a highly selective iNOS inhibitor (control: 754 +/- 184; L-NAME, 1 mM 329 +/- 87; 1400 W, 20 mM: 547 +/- 25; Ca2+ -free/EDTA: 490 +/- 184 cpm [3H]-citrullin/10(6) cells per 45 min, p < 0.05, n = 7-8). The data obtained demonstrate that frog urinary bladder mucosa epithelial cells provided antidiuretic hormone-induced increase of osmotic water permeability contain nitric oxide synthase. The presence of inducible (iNOS) as well as constitutive isoform(s) revealed in these cells allows to suggest involvement of NOS in intracellular signaling pathways regulated water transport across the epithelium.

Hydrobia ulvae (Gastropoda: Prosobranchia): a new model for regeneration studies.

Within 2 weeks of decapitation, Hydrobia ulvae was able to regenerate new head structures including buccal ganglia. It was also capable of regenerating propodial ganglia after anterior foot amputation. The functional regeneration of the buccal ganglia was demonstrated by behavioural observations and by electrophysiological experiments. The presence of the oesophagus was shown to be important for regeneration of the buccal complex. H. ulvae provides a new model for regeneration studies, so details of the topographic anatomy and biology of this species are described. To standardize experimental animals in future studies, the effects of age, sex and trematode infestation on the regeneration capacity of H. ulvae have been evaluated. The high capacity for regeneration together with the possibility of using electrophysiological techniques makes H. ulvae a favourable model in which to study neurogenesis in adult animals.

[Comparative morphofunctional analysis of the gastropod-trematode interactions]. / Sravnitel'nyi morfofunktsional'nyi analiz vzaimootnoshenii v sisteme molliusk-trematoda.

A morphofunctional aspect of pathogenesis in various mollusc--trematode combinations is very different. To evaluate the level of antagonism between the host and parasite, it is reasonable to use a summarized parameter of general physiological state of infected individuals. The somatic growth of the host could be used for that purpose. Analysis of original and reference data has shown, that the gastropod growth response to the trematode infection depends on the longevity of molluscs. The growth acceleration (gigantism) in infected individuals is common for snails with the intermediate longevity (3-4 yr), such as Hydrobia, Onobia and Bithynia (Prosobranchia: Rissoacea). As an exception, the gigantism is observed in the short-living species, which do not change the growth rate when they are parasitized: Biomphalaria, Bulinus, Helisoma and Lymnaea (Pulmonata: Basomathophora). Earlier, it was assumed that the single trend manifested in the infected long-living species (6-27 yr), such as Littorina (Prosobranchia: Littorinacea) and Cerithidea (Prosobranchia: Cerithiacea), in the decrease of the growth rate. The only case of the growth acceleration within the long-living molluscs was detected in the Littorina littorea from the White Sea infected with low pathogenic partenits of Cryptocotyle lingua (Heterophyidae). Based on the groups of snails with different longevity and being phylogenetically distant from each other, we can presume that the growth response to the infection correlates with the morphological peculiarities of hosts. The morphofunctional distinction in the system formed by trematodes and molluscs of different taxa are expressed in the parthenit localization in the host body and in the degree of digestive gland and/or gonad destriction. From the viewpoint of general physiological status of the host organism, the results of disfunctions in these organs are not similar. The destruction of digestive system ("energy power station" of organisms) is more fatal for infected individuals than a parasitic castration. Therefore, the localization of parasites in the mollusc gonad can be recognized as a parasitic adaptation to decrease the probability of a premature host's death and respectively to prevent the elimination of parasites. Phylogenetically distant gastropods with different life cycles use a diverse reproduction strategy, which implies a different proportion of gonad and digestive gland volume. Thus, snail species from different taxa are not similar in relation to the life space, which could be "granted" to the parasite without any risk to reduce a viability of the host. The space lack forces the parthenits parasitizing in the molluscs with the longer life span to occupy the digestive gland tissues, that increases their pathogenicity and decreases the host's growth rate.