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Petroleum water soluble fraction (WSF) impairs organisms, but damages may vary among cell and tissue levels. The aim of the present study was to evaluate the acute (24 h, 48 h, 72 h) and subchronic effects (36 days) of WSF (0%, 25% and 100%) in juveniles of the Neotropical top predator fish Hoplias aff. malabaricus. The effects of WSF were evaluated at a molecular level using the comet assay and micronucleus test for genome damage; and at a morphological level through histological identification of liver pathologic lesions. In both acute and subchronic exposure we found low levels of DNA damage (< 10% of comet tail) and non-significant frequency of micronucleus in WSF exposed fish. The most significant liver lesions in WSF exposed fish were fatty vacuolization, hypertrophy and focal necrosis. Since these tissue injuries were progressive and persistent, their irreversibility may negatively affect fish recruitment, even in a such resistant top predator.

The transmembrane domain (TM) and flanking regions of glycosyltransferases (GTs) have been implicated in the localization of these proteins in the Golgi apparatus (GA). alpha3/4 Fucosyltransferase III (FT3wt) (EC 2.4.1.65) is localized in the trans-Golgi and trans-Golgi network (TGN) of baby hamster kidney (BHK) cells and synthesizes Lewis determinants associated with cell adhesion events. We have evaluated the effect of removing the cytosolic domain on the localization of the enzyme and its capacity for synthesizing the Lewis A (Le A) determinant. The mutant where the cytoplasmic domain (Asp-2 to Trp-13) of FT3wt has been deleted (FT3dc) was localized in the Golgi but it was shifted to earlier compartments than FT3wt. The mutant was not detected on the plasma membrane (PM) and glycosylation analysis indicated that FT3dc was transported beyond the endoplasmic reticulum (ER) since complex type glycosylation was observed. Cells expressing FT3dc showed a significantly lower efficiency to synthesize Le A when compared with cells expressing FT3wt, in vivo. This reduction was not due to lower specific activity because both enzyme forms had a similar specific activity in vitro. Therefore, removal of FT3 cytosolic tail caused a shift in enzyme distribution to earlier Golgi compartments concomitant to the decrease of its biosynthetic capacity.

Fucosyltransferase III [galactoside 3(4)-L-fucosyltransferase; EC 2.4.1.65] (FT3) is a Golgi type II membrane protein that catalyses the synthesis of fucosylated Lewis motifs that are associated with cell-adhesion events and are differentially expressed during cell differentiation. In the present work, the full-length membrane bound form of FT3 has been expressed in baby hamster kidney cells. The enzyme has been found in the trans-Golgi and trans-Golgi network (TGN) of the transfected cells, where it appeared as monomers and dimers, but not as oligomers with high molecular masses. Therefore oligomerization is not the basis for correct localization of FT3 in the Golgi. The enzyme has been purified, with a final yield of 2% and a total purification of 2900-fold, by DEAE-Sepharose, SP-Sepharose, GDP-Fractogel and Superdex 200 chromatography. The purified enzyme showed a clear preference for the Gal beta 3GlcNAc motif in oligosaccharides conjugated with the hydrophobic tail (CH(2))(3)-NHCO-(CH(2))(5)-NH-biotin. Substitution of galactose with alpha 2-linked fucose or alpha 2,3-linked N-acetylneuraminic acid yielded a 1.9-fold increase or a 43% decrease in activity respectively. The enzyme showed no activity towards asialofetuin, a glycoprotein containing the Gal beta 3GlcNAc acceptor motif. Therefore it has been concluded that the membrane-bound form of FT3 is present in the Golgi and the TGN in an equilibrium of monomers<-->dimers, which might fucosylate glycans from glycolipids, but not from glycoproteins.