Alfredo Flury

Molecular cloning and expression of the β-hydroxysteroid dehydrogenase gene from Pseudomonas testosteroni

The structural gene (hsd) of the Pseudomonas testosteroni encoding the 17 beta-hydroxysteroid dehydrogenase has been cloned using the cosmid vector pVK102. Escherichia coli carrying recombinant clones of hsd, isolated by immunological screening, were able to express the biologically active enzyme, as measured by the conversion of testosterone into androstenedione. Subcloning experiments, restriction and deletion analysis, and site-directed insertion mutagenesis showed that the hsd gene is located within a 1.3-kb HindIII-PstI restriction fragment. A 26.5-kDa protein encoded by a recombinant plasmid containing this Ps. testosteroni DNA restriction fragment was detected by SDS-PAGE analysis of in vitro [35S]methionine-labeled polypeptides.

A novel activity for a group of sesquiterpene lactones: inhibition of aromatase.

A group of eleven sesquiterpene lactones isolated from different Asteraceae species from north‐western Argentina were investigated for their inhibitory action on the estrogen biosynthesis. Seven of them, of different skeleton types, were found to inhibit the aromatase enzyme activity in human placental microsomes, showing IC50 values ranging from 7 to 110 μM. The most active were the guaianolides 10‐epi‐8‐deoxycumambrin B (compound 1), dehydroleucodin (compound 2) and ludartin (compound 3). These compounds were competitive inhibitors with an apparent K i=4 μM, K i=21 μM and K i=23 μM, respectively. Compounds 1 and 2 acted as type II ligands to the heme iron present in the active site of aromatase cytochrome P450 (P450arom). Besides, all of them failed to affect the cholesterol side‐chain cleavage enzyme activity on human placental mitochondrias. This is the first report on the aromatase inhibitory activity of this group of natural compounds.

Regulation of the 3β-hydroxysteroid dehydrogenase activity in tissue fragments and microsomes from human term placenta: Kinetic analysis and inhibition by steroids

The effects of 50 microM of progesterone (P4), estradiol (E2), estrone (E1), estriol (E3), dehydroepiandrosterone (DHIA), androstenedione (delta 4) and testosterone (T) on the bioconversion of [3H]pregnenolone (6 nM) to [3H]P4 were investigated by incubating 200 mg of tissue fragments as well as equivalent aliquots of microsomes from human term placenta during 30 min. All the steroids assayed, except E3, significantly inhibited the [3H]P4 formation in a microsome incubation system with respect to the control assay (P less than 0.001). Conversely in a tissue incubation system. P4, E1 as well as E3 had no effect on [3H]pregnenolone bioconversion while E2 slightly decreased the [3H]P4 formation (P less than 0.05) compared with the control. A significant inhibition was observed in this system with the other steroids (P less than 0.001). To investigate these apparent different results of inhibition-noninhibition of the same steroids irrespective of the system of incubation used, the effects of P4, E2 and T on 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity were studied in tissue fragments and microsomes in kinetic terms. The results found indicate that these steroids inhibited in a competitive fashion the 3 beta-HSD activity in both systems. The different Ki values found in tissue fragments and microsomes respectively for P4 (1.8 microM vs 0.5 microM), E2 (2.3 microM vs 0.6 microM) and T (0.25 microM vs 0.3 microM) explain the bioconversion results obtained in presence of 50 microM of the same steroids. These results include inhibition of [3H]P4 formation by T in tissue fragments as well as in microsomes whereas P4 and E2 inhibited the [3H]P4 formation only in microsomes. Furthermore, the comparison of these Ki values with the available data of intraplacental and circulating concentrations of the same steroids in human term pregnancy suggest that only P4 would be expected to cause marked 3 beta-HSD inhibition in physiological conditions.

Nucleotide sequence of a pregnancy-specific β1 glycoprotein gene family member

The pregnancy-specific β1 glycoprotein (PSG) genes encode a group of heterogeneous proteins produced in large amounts by the human syncytiotrophoblast. Their expression seems to be regulated at the transcriptional level during normal pregnancy. In the present work, we isolated from a human placental library a 17 kb genomic fragment corresponding to a member of the PSG multigene family. DNA sequence analysis of 1190 nucleotides upstream of the translational start and of the first intron, revealed the presence of several putative regulatory sequences. In a transient chloramphenicol acetyltransferase expression assay, 5′ flanking sequences within 123 nucleotides upstream to the first major transcription initiation site, functioned as a strong promoter in COS-7 cells. Meanwhile, sequences 5′ further upstream had the ability to abolish this promoter activity. The sequence analyzed did not contain any obvious TATA-like boxes or G+C-rich regions, suggesting the existence of unique promoter elements implicated in transcription initiation and regulation of this PSG gene family member.

Effect of estradiol-17β on the conversion of pregnenolone to progesterone in human term placenta incubated in vitro

The effect of different doses of estradiol-17 beta (E2) on the metabolic pregnenolone to progesterone pathway in fragments of human term placenta incubated in vitro was studied. Doses considered as being physiological of 0.09 and 0.9 microM had a stimulatory effect on the conversion (p less than 0.008 to 0.016). However, a supraphysiological dose of 45 microM showed an inhibitory activity related to the maximal stimulation (p less than 0.03). A dose of 0.9 microM E2 favoured the accumulation of (3H)-progesterone in the tissue (p less than 0.05). These results suggest that E2 may regulate the synthesis of progesterone in human term placenta.

Topography of human placental 3β-fi-hydroxysteroid dehydrogenase/gD5-4 isomerase in microsomal membrane. A study using limited proteolysis and immunoblotting

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase Δ5-4 isomerase (3β-HSD) catalyzes the formation of Δ4-3-ketosteroids from Δ5-3β-hydroxysteroids in placental, adrenal, testicular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3β-HSD within the human placental microsome membranes employing immune-replica analysis in combination with surface specific proteolysis. The crucial domains of the enzyme for the dehydrogenase and isomerase reactions are inactivated by proteinase treatments under conditions where latency of hexose-6-phosphate dehydrogenase was 95%. The data indicate that these crucial domains face the cytosolic side of the endoplasmic reticulum membrane. Incubation of the intact microsomes with trypsin produces several immune reactive fragments ranging from 29 to 11 kDa in addition to 42 kDa native enzyme, one of them being shielded by the membrane structure and/or by other intrinsic and peripheral membrane proteins. Carboxypeptidase Y degraded the C terminus of the 42 kDa native 3β-HSD in intact and detergent-disrupted microsomes, preserving partially a fragment of 31 kDa. The results from the carboxypeptidase Y digestion indicate that the carboxy terminal end of the 3β-HSD enzyme is located on the cytoplasmic surface of the endoplasmic reticulum and that only a small fragment of approx. 11 kDa could be removed easily without affecting the enzyme activity. From these data and the predicted hydropathy analysis from the literature, we tried to assign a transmembrane arrangement to the human placental 3β-HSD. Our results support a topology model in which practically all the structural 3β-HSD enzyme is exposed to the cytoplasmic side of the membrane with one NH2-terminal-anchoring segment and all the 3β-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP1, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and α-mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP1 is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.

Translation in a cell-free system of mRNA from term placenta extracted by guanidine hydrochloride.

Human term placenta RNA and polyadenylated mRNA were prepared using guanidine HCl and oligo (dT)-cellulose affinity chromatography. Both fractions translated in a wheat germ cell-free system showed, under optimal condition of K+ and Mg++ ions and spermidine, about 9 times activity for RNA and 15–25 times for poly(A+) mRNA greater than the control. Homogenization of the tissue at high speed compared to that at low speed improved 4-fold activity.Analysis of tritiated products by SDS-polyacrylamide gel electrophoresis and detected by fluorography showed more than ten different intensity bands ranging between 12 and 66 kD. According to the results obtained, guanidine HCl is an advantageous procedure for the extraction of RNA from this nuclease-rich tissue compared with that obtained with phenol extraction, in both activity and in larger translation products.

Identification and molecular weight of SP1 synthesized from mRNA of human placenta in a wheat germ cell-free system

Poly (A+)-mRNA obtained from human term placenta using guanidine HCl and oligo (dT) cellulose chromatography was translated in a wheat germ cell-free system. SDS-polyacrylamide gel electrophoresis analysis of the translation products revealed the presence of several polypeptides with molecular weights ranging from 10 KD to 70 KD. A single protein band representing around 1% of the total radioactive proteins synthesized in the presence of 2.5 μg of mRNA was isolated by immunoprecipitation, using specific antiserum against either the native ‘Pregnancy-specific β1-glycoprotein’ or a reduced and carboxymethylated derivative. The molecular weight of 31–2 KD of this translation product corresponding to the nonprocessed precursor could account for the 43 KD value assigned to the protein purified from human pregnant serum.