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Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.

Background: DAX1 (NR0B1), a member of the nuclear receptors super family, has been shown to be involved in the genetic sex determination and in gonadal differentiation in several vertebrate species. In the aquaculture fish European sea bass, Dicentrarchus labrax, and in the generality of fish species, the mechanisms of sex determination and differentiation have not been elucidated. The present study aimed at characterizing the European DAX1 gene and its developmental expression at the mRNA level.

Trace metals, whether essential such as Cu and Zn or nonessential such as Cd, are taken up by aquatic invertebrates from both food and solutions. After ingestion, the metal is transported around the body, perhaps to be accumulated in particular target organs or even to be excreted (Rainbow, 1998). In most invertebrates, body concentrations of the non-essential metal Cd do not appear to be regulated (Rainbow, 1985;Amiard et al., 1987;Rainbow, 1998). Metal toxicity is often postulated to arise from reactions occurring in the cytosol, through nonspecific binding of the metal to non-thionein ligands, which are physiologically important molecules (metalloenzymes or small peptides such as glutathione) and are inactivated by metal binding (Mason and Jenkins, 1995). Control of intracellular metal toxicity and detoxification of accumulated trace metals is generally achieved via the production of metal-binding ligands that sequester metals. These cellular ligands can be found in the particulate fraction of the cells such as mineral deposits, granules or lysosomes (George, 1990;Mason and Jenkins, 1995) and in the cytosol such as metallothioneins (Engel and Roesijadi, 1987). Metallothioneins, a family of low molecular mass cysteine-rich proteins, have been shown to occur in most zoological taxa (Amiard and Cosson, 1997). However, in polychaetous annelids this type of protein has not yet been isolated.Nereis diversicolor (Hediste diversicolor, recent denomination) is a polychaete living in the mud of estuaries which, in Europe, is routinely contaminated by heavy metals. Previous work (Nejmeddine et al., 1988) showed that in animals exposed to Cd the metal was bound to two pools of proteins of high molecular mass (metalloprotein I, MPI, >67·kDa) and low molecular mass (MPII, of about 20·kDa). MPI is the extracellular hemoglobin of this annelid (Demuynck and Dhainaut-Courtois, 1993). The primary structure of MPII isolated from whole worms was different from that of a metallothionein, consisting of 119 amino acid residues with only one cysteine residue , but shares 80.8% identity with the myohemerythrin isolated by Takagi and Cox (1991) from the same species. MPII is a Cd-binding protein but is not a metallothionein. Metallothioneins are known to be induced by essential (Cu or Zn) and non-essential (Cd) metals and are thought to be important both in the detoxification of trace metals and in the metabolism of intracellular Cu and Zn, as in the scavenging of reactive oxygen species (Palmiter, 1998;Klaassen et al., 1999). However, MPII appeared to correspond to a myohemerythrinlike pigment, known to play a mainly respiratory role and with no known function of detoxification. Immunocytochemical studies showed that MPII is located in granulocytes I Isolated guts of Nereis diversicolor revealed the existence of a cadmium-binding protein, the MPII, belonging to the group of hemerythrins and myohemerythrins. The presence of MPII in the cells of the intestine was demonstrated by immunocytochemistry, using anti-MPII, a monoclonal antibod...