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Biomedical subjects

A Flury

Publications and source records attributed to A Flury.

At least 19 recordsLinked to original sources

A new death domain associated with gestational trophoblastic diseases induces apoptosis in distinct cell types.

Gestational trophoblastic diseases, like the complete hydatidiform mole (CHM), are a group of human interrelated neoplasms whose etiology and progression is poorly understood at the molecular level. We have previously reported the cloning and expression of a new tumor necrosis factor receptor (TNF-R) related transcript, named CHMS-1 that encodes a potential death domain. Here we show that ectopic expression of the putative CHMS-1 death domain specifically induced apoptosis in a dose-dependent manner, in trophoblastic (JEG-3) and non-trophoblastic (COS-7) cells. We also investigated the expression of apoptosis-related molecules such as Bcl-2 and p53 and demonstrated that Bcl-2 is repressed in CHM while p53 is overexpressed in CHM compared with persistent gestational trophoblastic tumors. Altogether, these data indicate that the CHMS-1 death domain is able to trigger apoptosis, thus suggesting that this new entity might be an important inducer of molar regression mechanisms in women.

Animals↗

Aromatase inhibition by an 11,13-dihydroderivative of a sesquiterpene lactone.

Compounds that inhibit aromatase activity are used for the treatment of breast cancer. A group of sesquiterpene lactones inhibit aromatase activity and also exert cytotoxicity through their reactive alpha-methylene-gamma-lactone group. To synthesize sesquiterpene lactones with greater specificity for aromatase inhibition and lower cytotoxicity, we chemically reduced the alpha-methylene-gamma-lactone group in the active aromatase inhibitor 10-epi-8-deoxycumambrin B (compound 1), to obtain the new compound 11betaH,13-dihydro-10-epi-8-deoxycumambrin B (compound 2). Reduction of the alpha-methylene-gamma-lactone group abrogated the cytotoxic activity of compound 1 against the JEG-3, HeLa, and COS-7 cell lines. Compound 2 had higher aromatase inhibitory activity than compound 1 (IC(50) = 2 +/- 0.5 microM versus 7 +/- 0.5 microM, K(i) = 1.5 microM versus 4.0 microM) and was a more potent type II ligand to the heme iron present in the cytochrome P450(arom) active site. Compound 2 inhibited aromatase activity in JEG-3 cells in a comparable manner to the inhibitor aminoglutethimide (AG) used clinically for the treatment of breast cancer. Additionally, compound 2 inhibited androstenedione-induced uterine hypertrophy in sexually immature mice (41% of uterine weight suppression for compound 2 versus 51% for AG). We conclude that the anti-aromatase activity of sesquiterpene lactones does not depend on the presence of the highly reactive alpha-methylene-gamma-lactone group, whereas their cytotoxicity does. These findings may facilitate the development of safer agents for breast cancer therapy.

Aminoglutethimide↗

[Is brain surgery on primates in basic research morally acceptable?]

In the contemporary controversy about the legitimacy of vivisection a few basic assumptions are shared by nearly all participants of the discussion. (I) Pure Research in the service of medicine is of great value for humankind. It contributes to prolonging human life and the alleviation and prevention of human suffering. (II) Brain surgery for the sole purpose of pure research is morally unacceptable in the case of any human being. (III) Primates are sensitive beings which lead a rich social life and are endowed with remarkable intellectual capacities. (IV) Primates have a moral standing, possibly to a lesser degree compared with human beings, certain acts are therefore an injustice toward them. The controversy then is about the question whether premise (I) outweighs (IV), i.e. whether the benefit of the pure research is from a moral point of view more important than the suffering of innocent primates. I shall present four arguments against such a conclusion. 1) According to premise (I) brain surgery on human beings for the sole purpose of pure research is morally unacceptable. Since this prohibition is meant to include all human beings it cannot rest on the exclusive human possession of reason because e.g. some mentally handicapped human beings lack this faculty. All other properties which may be named as basis for the ascription of a moral status which forbids brain surgery for pure research, are possessed also by some animals, especially primates; therefore it is impossible to deny them the same moral status. 2) Brain surgery on primates is confronted with an insoluble dilemma: If the characteristics of the primate brain are very similar to that of human beings, the scientific benefit is obvious, but the procedure appears to be morally unacceptable exactly because of this similarity. If, on the other hand, the characteristics differ significantly, brain surgery may seem legitimate but the scientific benefit becomes doubtful at best. 3) We could quite easily save hundreds of human lives if e.g. speed limits would be reduced (say) by half. Most of us, however, are unwilling to accept such a loss of quality of life in order to save a certain number of human lives. Since we are no prepared to pay this comparatively modest price, we have, in my eyes, no moral right to impose considerable pain and suffering on a primate to save human lives. 4) Pure research in the service of human medicine is from a moral point of view of great importance. Since most of the work in this area is done or financed by private corporations and not by state institutions, from a economical point of view the aim consists in making profit. Since the latter aspect has gained more and more weight in the last years the moral worth of pure research cannot rule out any other moral concern.

Journal Article↗

Differential expression of a tumor necrosis factor receptor-related transcript in gestational trophoblastic diseases in women.

Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.

Amino Acid Sequence↗

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 microM. 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 Ki = 4 microM, Ki = 21 microM and Ki = 23 microM, 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.

Anti-Ulcer Agents↗

A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene.

We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.

Amino Acid Sequence↗

Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types.

Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.

Binding Sites↗

Topography of human placental 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase in microsomal membrane. A study using limited proteolysis and immunoblotting.

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase delta 5-4 isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids in placental, adrenal, testicular and ovarian tissues. In the present study was investigated the transverse-plane topography of 3 beta-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 3 beta-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 3 beta-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 3 beta-HSD. Our results support a topology model in which practically all the structural 3 beta-HSD enzyme is exposed to the cytoplasmic side of the membrane with one NH2-terminal-anchoring segment and all the 3 beta-HSD enzyme activity facing to the cytoplasmic side within the 31 kDa NH2-terminal peptide.

Carboxypeptidases↗

Low aromatase activity in microsomes from complete hydatidiform mole.

The complete hydatidiform mole (CHM) is characterized by the presence of aberrant placenta, with hyperplasia of cyto- and syncytiotrophoblasts and the absence of maternal genetic information. Steroidogenesis in this condition is, thus, of special interest. In this study we investigated the kinetic parameters of aromatase in microsomes from CHM compared with those in normal early placenta (NEP). The enzyme activity was determined by measuring the conversion of [3H] testosterone to [3H]estradiol plus [3H]estrone. The Km value for testosterone was 33 nmol/L in CHM and 17 nmol/L in NEP of similar gestational ages. Aminoglutethimide, a nonsteroidal inhibitor, decreased in a dose-dependent manner and with the same potency the aromatization of testosterone in both tissues (ID50, 2 vs. 1 mumol/L in CHM and NEP, respectively). These results suggest that the enzymes from the two sources are kinetically similar. However, the enzyme efficiency, expressed as the maximum velocity/Km ratio, was 17-fold lower in CHM than in NEP tissue (1.22/33 vs. 10.68/17 min/mg.mL). These findings suggest that in molar pregnancy the decreased capacity of trophoblast tissue for the formation of estrogen could increase the testosterone concentration inside the molar vesicle, which, in turn, as we previously reported, inhibits progesterone formation. All of these data could provide an explanation for the low circulating level of progesterone, which may directly or indirectly affect the spontaneous expulsion of this aberrant tissue in the second trimester of pregnancy.

Abortion, Therapeutic↗

Nucleotide sequence of a pregnancy-specific beta 1 glycoprotein gene family member. Identification of a functional promoter region and several putative regulatory sequences.

The pregnancy-specific beta 1 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.

Amino Acid Sequence↗

Molecular cloning and expression of the beta-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.

17-Hydroxysteroid Dehydrogenases↗

Kinetic analysis of 3 beta-hydroxysteroid dehydrogenase activity in microsomes from complete hydatidiform mole.

Microsomes isolated from complete hydatidiform moles (CHM) were able to convert [3H]pregnenolone to [3H]progesterone which indicates the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity. The kinetic parameters found (Km = 0.63 microM and Vmax = 1-3.05 nmol/min/mg of protein) were like those observed in microsomes from normal early placenta (NEP) of similar gestational age (herein) and term placenta suggesting that the enzymes from the three sources are kinetically similar. Testosterone, progesterone and estradiol in a dose range of 0.05-5 mumol/l inhibited differently the in vitro conversion of [3H]pregnenolone to [3H]progesterone in a dose-dependent manner. The steroid concentrations necessary to inhibit the conversion of pregnenolone to progesterone by 50% (ID50) in CHM were 0.1 microM for testosterone, 0.6 microM for progesterone and 3 microM for estradiol, whereas in NEP they were 2.5, 1 and 5 microM respectively. The Ki values calculated from these ID50 in CHM together with the reported levels of endogenous steroids indicate that the accumulation of testosterone and progesterone inside the molar vesicle could physiologically regulate the rate of further conversion of pregnenolone to progesterone. The present findings could provide an explanation for the low level of progesterone in patients with CHM in the second trimester of pregnancy which in turn may directly or indirectly affect the spontaneous expulsion of this aberrant tissue.

Binding, Competitive↗

Regulation of the 3 beta-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.

3-Hydroxysteroid Dehydrogenases↗

Expression of pregnancy specific beta 1-glycoprotein gene in human placenta and hydatiform mole.

Transcriptional studies of the placental protein Pregnancy Specific beta 1-Glycoprotein (SP1 or PS beta G) gene with a cDNA probe in Northern blot analysis showed 15-20 folds mRNA increase in term placenta compared with early placenta and hydatiform mole. This value parallels the SP1 amount translated in wheat germ cell-free system. We conclude that SP1 biosynthesis is regulated at transcriptional level during placental development and a similar mechanism would occur in hydatiform mole which is a hyperplastic trophoblast tumor.

Bacteriophage lambda↗

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 alpha-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.

Cell-Free System↗

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 micrograms of mRNA was isolated by immunoprecipitation, using specific antiserum against either the native 'Pregnancy-specific beta 1-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 form human pregnant serum.

Cell-Free System↗

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.

Animals↗

Effect of estradiol-17 beta 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.

Dose-Response Relationship, Drug↗