Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase.

The cuticle of parasitic nematodes consists primarily of a network of collagen molecules. The enzyme responsible for collagen maturation is prolyl 4-hydroxylase, making this enzyme a central activity in cuticle biosynthesis and a potentially important chemotherapeutic target. Adult and embryonic Brugia malayi are shown to be susceptible to inhibitors of vertebrate prolyl 4-hydroxylase, with exposed parasites exhibiting pathologies consistent with a disruption in cuticle biosynthesis. A full-length cDNA (Ov-phy-1) encoding a catalytically active alpha-subunit of Onchocerca volvulus prolyl 4-hydroxylase was isolated and characterized. The derived amino acid sequence of Ov-phy-1 encoded a peptide that was most similar to the two Caenorhabditis elegans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vertebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame contained 11 introns, similar in structure to the gene encoding human prolyl 4-hydroxylase isoform I. Genomic Southern blot, EST and genomic PCR studies demonstrated that the O. volvulus genome contained between three and eight genes closely related to Ov-phy-1. Co-expression of Ov-phy-1 with the O. volvulus homologue of protein disulfide isomerase in a baculovirus system resulted in the production of enzymatically active O. volvulus prolyl 4-hydroxylase. In vitro production of enzymatically active O. volvulus prolyl 4-hydroxylase should facilitate identification of specific inhibitors of the parasite enzyme.

Two novel antifibrotics, HOE 077 and Safironil, modulate stellate cell activation in rat liver injury: differential effects in males and females.

The perisinusoidal stellate cells of the liver in an injury milieu undergo activation, acquiring a myofibroblast-like phenotype. In this state, they are the principal source of collagen and related proteins in fibrosis. The present studies evaluate the mechanism of action of two novel antifibrotic compounds, HOE 077 and Safironil, which were designed as competitive inhibitors of collagen protein synthesis. Fibrosis was induced in rats by administration of carbon tetrachloride, and activation was monitored as the level of collagen I mRNA or smooth muscle alpha-actin. Both male and female rats were studied. Stellate cell activation, rather than collagen synthesis, proved to be the target of both HOE 077 and Safironil in the intact liver. In culture, the drugs not only prevented the activation of stellate cells but also accelerated their deactivation. They were no more effective in co-cultures containing hepatocytes than in pure stellate cell cultures, indicating that metabolic conversion of HOE 077 was not required. Interestingly, the response of cells from females was greater than that of male cells, leading to the conclusion that stellate activation is sexually dimorphic. This finding may be relevant to the observation that fibrosis in chronic viral hepatitis progresses less rapidly and that hepatocellular carcinoma is less frequent in females than in males.