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Biomedical subjects

S Zoppi

Publications and source records attributed to S Zoppi.

At least 19 recordsLinked to original sources
PubMed1994

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo.

Androgen-Insensitivity Syndrome↗
PubMed1994

Effect of the in vivo treatment with the synthetic steroid 17 beta-methoxy-17-methyl-(5 alpha)-1 H'-androstane-(3,2-c) pyrazole (17MM) on the in vitro formation of DHT in the epididymis of the rat.

Adult male Sprague-Dawley rats were injected subcutaneously (s.c.) for 21 days either with the synthetic steroid 17 beta-methoxy-17-methyl-(5 alpha)-1 H'-androstane-(3,2-c) pyrazole (17MM) at two dose levels (0.1 and 1 mg/kg/day) or with the vehicle used to dilute the steroid. At autopsy, caput epididymes and ventral prostates were removed and incubated in vitro. The effect on the 5 alpha-reductase (5 alpha-R) activity was evaluated by measuring the amounts of DHT formed from 14C-testosterone (14C-T) used as the substrate. The in vivo administration of 17MM at the dose of 0.1 mg/kg/day resulted in a significant decrease in the formation of DHT at the level of the epididymis, while there was no effect on the prostate. The dose of 1 mg/kg/day did not induce any modification in the formation of DHT in either organ. The determination of serum LH showed that the treatment with either dose did not alter serum LH titers. These findings suggest that the in vivo treatment with 17MM lowers the in vitro formation of DHT from T at the epididymal, but not at the prostatic level and consequently suggests a selective inhibitory effect of 17MM on the 5 alpha-R present in the epididymis.

5-alpha Reductase Inhibitors↗
PubMed1992

[Mirizzi's syndrome type II: a diagnostic and therapeutic problem].

The paper reports a case of cholestatic icterus caused by a large calculus in the infundibulum of the gallbladder with a cholecysto-choledochal fistula and hepato-choledochal block (Mirizzi's type II syndrome). The diagnostic iter complicated the evolution, raising questions regarding the therapeutic strategy.

Biliary Fistula↗
PubMed1992

Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli.

We have expressed fusion proteins encoding defined segments of the coding segment of the human androgen receptor (hAR) in Escherichia coli using the pGEX-2T expression vector. Large quantities of fusion proteins containing glutathione-S-transferase (GST) linked to the amino or carboxy terminal region of the receptor and a fusion protein containing the complete amino acid sequence of the androgen receptor were produced in soluble form. The GST hAR fusion proteins containing the hormone-binding domain of the androgen receptor exhibit high affinity specific binding for a variety of natural and synthetic androgens. Analysis of the binding properties of the complete and truncated androgen receptor fusion proteins revealed that the amino terminus affects the Kd of the fusion proteins for mibolerone (0.89 vs. 3.43 nM for the truncated and complete fusion proteins, respectively). Despite these differences, both the truncated and complete hAR fusion proteins exhibit a higher affinity for dihydrotestosterone than for testosterone, implying that the preferential affinity for dihydrotestosterone observed in androgen receptor prepared from native sources is a measure of the inherent structure of the hormone-binding domain. Furthermore, the ligand-receptor complex is stable, as the ligand is not easily displaced with unlabelled competitor and is stable to mild heat denaturation. Fusion proteins containing the DNA-binding domain demonstrate specific DNA binding, as evidenced by studies using segments of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) and synthetic glucocorticoid response elements. These studies establish that GST hAR fusion proteins exhibit physical properties similar to those of native androgen receptor. Affinity purification using a glutathione affinity resin and cleavage of the fusion proteins at a thrombin cleavage site permits a marked enrichment using a two-step purification. The use of such methods will facilitate the study of the normal and mutant receptor proteins.

Base Sequence↗
PubMed1992

Selective inhibition of the 5 alpha-reductase of the rat epididymis.

The effect of several synthetic steroids belonging either to the 4-aza-3-oxo-steroid family or to androstene and androstane derivatives was investigated "in vitro" on the epididymal as well as prostatic 5 alpha-reductase activity. For this purpose rat caput epididymis and prostate were incubated with the different steroidal compounds at molar concentrations of 10(-7), 10(-6), and 10(-5) in the presence of labelled testosterone as substrate. The steroids 4-MA (17 beta, N,N-diethyl-carbamoyl-4-aza-5 alpha-androstan-3-one) and 4-OH-A (4-hydroxy-androstenedione), already known to be effective 5 alpha-reductase inhibitors at the level of the prostate, have been used as reference molecules. The 5 alpha-reductase activity was evaluated by measuring pg of dihydrotestosterone (DHT) formed in 2 h of incubation by mg of tissue. The steroids A, B, C, F, G and I inhibit the formation of DHT in the rat epididymis although to different extents; they are also equally effective on the formation of DHT in the rat prostate. The steroids D, E, H and L are devoid of any inhibitory property on the formation of DHT in both the rat epididymis and prostate. The most interesting results were obtained with compound M which exhibits a dose-dependent and significant inhibitory effect on the formation of DHT in the epididymis, but it is inactive at the level of the prostate. These findings suggest that it is possible (a) to selectively interfere with the 5 alpha-reductase of the epididymis without affecting that present in the prostate, and (b) consequently to envisage new ways to regulate male fertility.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1992

Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene.

We have analyzed the nucleotide sequence of the androgen receptor from 22 unrelated subjects with substitution mutations of the hormone-binding domain. Eleven had the phenotype of complete testicular feminization, four had incomplete testicular feminization, and seven had Reifenstein syndrome. The underlying functional defect in cultured skin fibroblasts included individuals with absent, qualitative, or quantitative defects in ligand binding. 19 of the 21 substitution mutations (90%) cluster in two regions that account for approximately 35% of the hormone-binding domain, namely, between amino acids 726 and 772 and between amino acids 826 and 864. The fact that one of these regions is homologous to a region of the human thyroid hormone receptor (hTR-beta) which is a known cluster site for mutations that cause thyroid hormone resistance implies that this localization of mutations is not a coincidence. These regions of the androgen receptor may be of particular importance for the formation and function of the hormone-receptor complex.

Amino Acid Sequence↗
PubMed1992

Immunoreactive androgen receptor expression in subjects with androgen resistance.

Individuals with androgen resistance encompass a spectrum of phenotypic abnormalities ranging from complete testicular feminization to undervirilized men. Such subjects have been classified according to the hormone-binding characteristics in genital skin fibroblasts and on the basis of the mutation in the androgen receptor (AR) gene. Antibodies to the amino-terminal region of the human AR were used to develop an immunoblot assay for the comparison of androgen binding with the amount of AR expressed in genital skin fibroblasts. In controls and 4 androgen-resistant subjects with DNA-binding domain mutations, levels of immunoreactive AR correlated closely with androgen-binding capacity. In 15 androgen-resistant subjects with qualitatively abnormal AR, immunoreactive AR levels tended to be higher than predicted from the ligand-binding capacity. Discordance between immunoreactivity and androgen binding also occurred in fibroblasts from 3 other subjects. One carries a stop codon in the AR gene and produces a truncated AR that is immunoreactive but does not bind androgen. Two carry single point mutations in the hormone-binding domain and produce immunoreactive AR that is normal in size but does not bind androgen.

Androgens↗
PubMed1992

Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance.

In some subjects with genetic and endocrine evidence of androgen resistance, no defect is demonstrable in the binding of androgen to its receptor in cultured genital skin fibroblasts. We have defined the molecular defect in the androgen receptor in four unrelated subjects in this category (termed receptor positive) with the phenotype of compete or incomplete testicular feminization. In these patients we detected amino acid substitutions in either exon 2 or exon 3, which encodes the DNA-binding domain of the androgen receptor. In one patient with incomplete testicular feminization, two separate mutations were present in exon 3. Introduction of these amino acid substitutions into the androgen receptor-coding segment leads to the expression of receptor proteins that bind ligand in a normal fashion but do not activate the transcription of the androgen-responsive mouse mammary tumor virus promoter. Mobility shift assays using androgen receptor fusion proteins produced in E. coli indicate that these mutations impair binding of the receptor to specific DNA sequences. In the subject with incomplete testicular feminization, a Ser-Gly substitution at amino acid residue 595 is able to partially restore DNA-binding activity to a mutant receptor protein that carries an Arg-Pro substitution at position 615. These findings indicate that mutations in amino acid residues crucial to the binding of the androgen receptor to target DNA sequences are a common cause of receptor-binding positive androgen resistance and that variable impairment of DNA binding can lead to distinctive phenotypes.

Adolescent↗
PubMed1991

A mutation in the DNA-binding domain of the androgen receptor gene causes complete testicular feminization in a patient with receptor-positive androgen resistance.

Androgen resistance is associated with a wide range of quantitative and qualitative defects in the androgen receptor. However, fibroblast cultures from approximately 10% of patients with the clinical, endocrine, and genetic features characteristic of androgen resistance express normal quantities of apparently normal androgen receptor in cultured genital skin fibroblasts (receptor-positive androgen resistance). We have analyzed the androgen receptor gene of one patient (P321) with receptor-positive, complete testicular feminization and detected a single nucleotide substitution at nucleotide 2006 (G----C) within the second "zinc finger" of the DNA-binding domain that results in the conversion of the arginine residue at position 615 into a proline residue. Introduction of this mutation into the androgen receptor cDNA and transfection of the expression plasmid into eukaryotic cells lead to the synthesis of a receptor protein that displays normal binding kinetics but is inactive in functional assays of receptor activity. We conclude that substitution mutations in the DNA-binding domain of the androgen receptor are one cause of "receptor-positive" androgen resistance.

Androgen-Insensitivity Syndrome↗
PubMed1991

Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg----Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function.

Analysis of the nucleotide sequence of the coding segment of the androgen receptor gene in a patient (N105) with the receptor-negative form of complete testicular feminization has revealed a single substitution (CGC----TGC) at nucleotide 2476. This alteration results in the conversion of an arginine at amino acid 772 to a cysteine. Introduction of this mutation into an androgen receptor cDNA and transfection of the mutant cDNA into COS cells result in the production of a receptor protein with an alteration in the apparent Kd of ligand binding (3 nM) compared to that of the normal androgen receptor (0.5 nM). The mutant receptor protein predicted for patient N105 also demonstrates thermal instability of ligand binding that is not associated with quantitative or qualitative changes in the immunoreactive androgen receptor protein. When assayed in cotransfection experiments using a mouse mammary tumor virus-chloramphenicol acetyl transferase reporter system, the N105 receptor protein appears to be about a tenth as active as the control receptor. These functional characteristics do not appear sufficient to account for the phenotype of complete testicular feminization and do not explain the profound deficiency of androgen receptor in cultured skin fibroblasts. Quantitative S1 nuclease protection assays reveal that the level of androgen receptor mRNA in fibroblasts from patient N105 is markedly reduced. These results suggest that the phenotype in patient N105 is due to two effects of the nucleotide substitution at residue 2476: the replacement of a crucial amino acid (772) in the hormone-binding domain that impairs the function of any receptor molecules formed and a decrease in the level of androgen receptor mRNA.

Androgen-Insensitivity Syndrome↗
PubMed1991

[Arterial diseases of surgical importance. Long-term control of diet-related risk factors].

From 1980 to 1990 a yearly medical-surgical control was carried out on 950 arteriopathic patients hospitalised in the General and Cardiovascular Surgery Institute, Milan University, during the above mentioned period. The operations for surgical reconstruction were mainly for arteriosclerotic lesions involving the abdominal-peripheral district (750 patients, 79%) and the supra aortic trunks (200 patients, 21%). 84% of the patients were males. Among the patients on follow-up, a relatively modest number (100 patients) did not attend the prescribed diet therapies for several reasons, consequently these patients have been considered as a control group. The comparison, between the patients who attended and those who did not attend the diet, has been made on the basis of the following parameters: cholesterolemia (COL), LDL, HDL, triglyceridemia (TG), VLDL, glycemia (GLI), body mass index (BMI) and arterial pressure (PA). The results have shown a decrease in the absolute normal values of cholesterol, LDL and triglycerides, while they have not shown the same significant variations regarding glycemia and body weight. In accordance with the literature, the diet seems to have obtained satisfactory results, especially regarding the values of lipidemia, with a reduction in the atherogenic risk index. Regarding smoke as a risk factor it has been shown that it is present in 80% of the patients at the beginning: 60% of the patients stop smoking at the moment they are hospitalised and the remaining 20% continue smoking. The hygienic-dietetic intervention is confirmed, also in our experience, as a curative, or even more, a preventive tool against the worsening of an already overt arteriosclerosis as well as a necessary support for drug therapies.

Arteriosclerosis↗
PubMed1990

Characterization of basolateral membrane Na/H antiport in rat jejunum.

Na uptake studies were performed in order to examine the activity of a Na/H exchanger in basolateral membrane vesicles isolated from rat jejunum. Experiments were carried out under voltage-clamped conditions in order to avoid electrodiffusional ionic movements. 1 mM Na uptake was found to be enhanced by an outward proton gradient and its initial rate was further increased by the presence of monensin or nigericin. The pH gradient-driven Na uptake was inhibited by 2 mM amiloride and unaffected by 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. The initial rate of the proton gradient-induced Na uptake was saturable with respect to external Na, with a Km of 13.6 +/- 1.4 mM and a Vmax of 35.4 +/- 2.2 nmol/mg protein per min. Li competed with Na for the exchange process, whereas K, Rb, Cs, tetramethylammonium had no effect. We conclude that rat jejunal basolateral membrane contains a Na/H exchanger whose properties are similar to those of the antiporter identified in the brush-border membrane.

Amiloride↗
PubMed1990

Aging and ATPase activities in rat jejunum.

In addition to the well-known (Na,K)-ATPase activity, an ouabain-insensitive Na-ATPase has been evidenced in the basolateral membrane of intestinal and renal cells from different mammals. Basolateral membranes of jejunal enterocytes from rats of different ages, i.e., very young, young, adult and old were separated by self-orienting, Percoll-gradient centrifugation. The total protein content and both Na- and (Na,K)-ATPase activities in initial homogenate and final pellets were analyzed. The dry weight of homogenate and pellet was also determined. The two ATPase activities and the protein content of the basolateral membrane fraction decrease with age when referred to the dry weight of the pellet. This diminution is also evident in the initial homogenate. The activation curve of Na-ATPase, hyperbolic in shape, gives Km and Vmax values unaffected by aging. The same behaviour is true for the kinetic parameters of (Na,K)-ATPase, which has a sigmoidal velocity curve. From these results, it seems that both Na- and (Na,K)-ATPase have the same characteristics in the basolateral membrane of the enterocyte throughout the life span of the animal, but they decrease quantitatively with aging.

Adenosine Triphosphatases↗
PubMed1989

Effect of neonatal estrogenization on testosterone metabolism in the prostate and in the epididymis of the rat.

The present experiments were performed in order to analyze whether the administration of estrogens (single injection of 500 micrograms of estradiol benzoate s.c.) to neonatal male rats might modify the weight of the ventral prostate and the epididymis as well as the metabolism of testosterone in these two organs. The metabolism of testosterone was evaluated in vitro using 14C-radiolabelled testosterone as the substrate. The metabolites dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), androstenedione, 5 alpha-androstane-3,17-dione (5-A-dione) and 3 alpha-hydroxy-5 alpha-androstane-17-one (androsterone) were quantified. After neonatal estrogen administration animals were killed on days 22 and 90 of age. The following changes were observed: (1) the body weight, the weight of the testes and of the ventral prostate were lower than in controls on both day 22 and 90; (2) the weight of the epididymides was higher than in controls on day 22 and lower on day 90; (3) in the ventral prostate the in vitro formation of DHT was lower and that of the diols was higher than in control tissue on day 22 of age; (4) the in vitro formation of alpha-reduced metabolites of the 17-keto series (5 alpha-A-dione + androsterone) was higher in ventral prostate of treated animals than in that of controls on day 22; (5) in treated animals, no formation of DHT in the caput epididymis was observed at day 22. On the contrary, at the same age the formation of androstenedione was higher than in controls; on day 90 of age the formation of DHT, androstenedione and the 5 alpha-reduced metabolites of the 17-keto series was identical in caput epididymis of the treated animals and of the controls, while the formation of the diols was higher in the treated than in the controls. The data indicate that neonatal estrogenization may induce important changes in testosterone metabolism in the prostates and in the epididymides of the rat.

Aging↗
PubMed1989

Three-month treatment with a long-acting gonadotropin-releasing hormone agonist of patients with benign prostatic hyperplasia: effects on tissue androgen concentration, 5 alpha-reductase activity and androgen receptor content.

The intraprostatic concentrations of testosterone (T) and dihydrotestosterone (DHT) have been measured in only a few men. We measured, in prostatic tissue obtained at surgery from seven men with benign prostatic hyperplasia, the effects of 3-month treatment with a long-acting GnRH agonist on 1) the intraprostatic concentrations of T, DHT, and 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha-diol); 2) prostatic 5 alpha-reductase activity; and 3) the prostatic content of androgen receptors (AR). Plasma T, DHT, and 3 alpha-diol levels also were measured. Prostatic tissue samples obtained at surgery from a group of untreated men with benign prostatic hyperplasia also were studied. The mean DHT and 3 alpha-diol concentrations in the prostatic tissue of the treated men were about 10% of those in untreated men (n = 19; P less than 0.01 for DHT and P less than 0.05 for 3 alpha-diol), and the mean intraprostatic T concentration in the treated men was about 25% of that in the control group (0.10 greater than P greater than 0.05). The mean in vitro formation of DHT by the prostatic tissue of the treated men was about 50% lower (P less than 0.05) than that by prostatic tissue of the untreated men (n = 9). The mean cytosolic AR content in the prostatic tissue of the treated men was significantly higher (P less than 0.05), whereas the mean nuclear content of both salt-extractable and salt-resistant AR was significantly lower (P less than 0.05) than that in the prostatic tissue of the untreated men (n = 8). The mean plasma T levels in treated men decreased from 4.77 +/- 1.79 (SD) ng/mL (16.5 +/- 6.2 nmol/L) to 0.27 +/- 0.42 ng/mL (0.9 +/- 1.5 nmol/L) after 1 month of therapy and remained in the castrate range thereafter. We conclude that pharmacological castration resulting from 3-month treatment with a long-acting GnRH agonist decreases the intraprostatic T concentration to about one fourth and those of DHT and 3 alpha-diol to about one tenth of the levels in untreated men. Thus, GnRH agonist treatment may not completely abolish intraprostatic androgen concentrations in metastatic prostatic cancer patients. The decrease in prostatic 5 alpha-reductase activity as well as the decrease in nuclear receptors are probably secondary to the decrease in plasma T concentrations.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1988

Antihormonal activities of 5 alpha-reductase and aromatase inhibitors.

The problem of developing androgen antagonists has been tackled so far only by synthesizing steroids able to displace testosterone and other androgens from their specific receptor sites. The observation that testosterone has to be converted intracellularly either to 5 alpha-reduced metabolites (DHT, 3 alpha-diol, etc.) or to estrogens, in order to become fully active on androgen-dependent structures (both central and peripheral), has opened the possibility of creating molecules which prevent these conversions, and which could then block the actions of testosterone. The availability of these new compounds has allowed a better understanding of the selective physiological role of each of the metabolites of testosterone, and to provide the basis for the development of new hormone antagonists to be used in those clinical conditions for which an inhibition of the actions of testosterone is foreseen. The usefulness of these enzyme inhibitors is underlined by some examples described in this paper. The results obtained may permit the formulation of the following conclusions: (1) The conversion of testosterone to its 5 alpha-reduced metabolites occurring in the neuroendocrine structures may represent an essential step for the appearance of the inhibitory feedback effect testosterone exerts on LH secretion; (2) Testosterone exhibits its negative feedback effect on FSH secretion as such and not following the local aromatization to estrogens; (3) Testosterone exerts its effect on the intrahypothalamic stores of LHRH acting as such and not following its local conversion either to 5 alpha-reduced metabolites or to estrogenic molecules; (4) Some of the new enzyme inhibitors (e.g. 4-OH-A) may represent an interesting tool for the treatment and/or the prevention of BPH and possibly of other androgen-dependent diseases (prostate carcinoma, acne etc.), as shown by their ability to prevent the in vitro conversion of testosterone to its 5 alpha-reduced metabolites both in the normal prostate of the rat and in the human BPH tissue.

Animals↗