Cloning and analysis of a cDNA encoding a putative serine protease comprising two trypsin-like domains of Trichinella spiralis.

The cDNA encoding a putative serine protease, TsSerP, was cloned by degenerative polymerase chain reaction and screening of the cDNA library from Trichinella spiralis adult-newborn larvae stage. Sequence analysis revealed the presence of two trypsin-like serine protease domains flanking a hydrophilic domain, with the catalytic triad residue histidine in the alpha domain substituted by an arginine residue. Southern blots indicated that this was a single copy gene in the parasite genome. Northern blots demonstrated a single 2.3-kb transcript during the muscle larvae and adult stages of T. spiralis. The recombinant protein from the TsSerP beta domain (betaSerP) was produced but not recognised by T. spiralis-infected swine serum. An anti-betaSerP polyclonal serum detected a 69-kDa polypeptide in the soluble antigens of T. spiralis muscle larvae. Immunolocalisation analysis located TsSerP on the inner layer of the cuticle and oesophagus of the parasite, suggesting a potential role in its moulting and/or digestive functions.

Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization.

The cytochrome P4507B1 (P4507B1) is responsible for the 7alpha-hydroxylation of dehydroepiandrosterone (DHEA) and other 3beta-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues.

Antioxidant effects of dehydroepiandrosterone and 7alpha-hydroxy-dehydroepiandrosterone in the rat colon, intestine and liver.

This study examined in healthy male Wistar rats the in vivo antioxidant effect of dehydroepiandrosterone (DHEA) and 7alpha-hydroxy-DHEA administered by intraperitoneal injections (50 mg/kg body weight) for 2 or 7 days. Markers of oxidative damage to lipids (thiobarbituric acid-reacting substances, TBARS) and to proteins (protein carbonyls) were assessed in colon, small intestine, and liver homogenates. DHEA and 7alpha-hydroxy-DHEA caused a decrease in body weight. DHEA treatment significantly increased liver, colon, and small intestine cell weights. After 7 days, DHEA exerted an antioxidant effect in all organs studied. In the colon, oxidative damage protection was accompanied by a goblet cell proliferation and increase in acidic mucus production. After 2 days, the antioxidant effect of 7alpha-hydroxy-DHEA was mainly observed in the liver. Nonprotein sulfhydryl groups (mostly glutathione levels) were altered by DHEA in the liver whereas they remained unchanged after 7alpha-hydroxy-DHEA treatment. The results indicate that in healthy animals, DHEA exerts a protective effect, particularly in the colon, by reducing the tissue susceptibility to oxidation of both lipids and proteins. This effect was not limited to a specific tissue, whereas the metabolite 7alpha-hydroxy-DHEA exerted its antioxidant effect towards the two markers of oxidative damage earlier than DHEA, and mainly in the liver.

Use of bioconversion for the preparation of [4-14C]-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone.

The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1. Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of [4-14C]-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.