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Biomedical subjects

G Romalo

Publications and source records attributed to G Romalo.

At least 19 recordsLinked to original sources
PubMed2003

Genital skin fibroblasts (GF) of patients with androgen insensitivity syndrome express higher insulin-like growth factor binding protein (IGFBP)-2, -3 and -5 than GF of normally virilized males.

BACKGROUND: We investigated the effects of androgens, estradiol (E2) and insulin-like growth factor (IGF)-I on IGF-II, insulin-like growth factor binding protein (IGFBP)-2, -3 and -5 and mRNA in genital fibroblasts (GF) from patients with complete androgen insensitivity (CAIS) and normally virilized males (C). METHODS: Proteins were measured by specific RIA and Western ligand blot, and specific mRNA levels by RT-PCR normalized by GAPDH levels. RESULTS: Secretion of IGF-II was lowered in CAIS (p<0.001) GF and by testosterone + IGF-I in C GF. Secretion of IGFBP-2 was higher (p<0.001) in CAIS GF and IGFBP-2 mRNA levels were increased by E2 in C GF (p<0.05). E2 stimulated IGFBP-2, -3 and -5 expression in CAIS GF. CAIS GF also secreted more IGFBP-3 (p<0.001) and accumulated 3-5 times more IGFBP-5 mRNA than C GF (p<0.001). CONCLUSION: In contrast to C GF, the availability of IGF-II in CAIS GF is apparently decreased by two facts: by the decreased expression and by increased expression of IGFBP-2, -3 and -5. Furthermore, E2 and IGF-I modulate the expression of IGF-II and IGFBP in GF. This may play a role in the failure to develop male external genitals in CAIS patients.

Androgen-Insensitivity Syndrome↗
PubMed2001

Decreased expression of IGF-II and its binding protein, IGF-binding protein-2, in genital skin fibroblasts of patients with complete androgen insensitivity syndrome compared with normally virilized males.

The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 +/- 9.7 vs. 106.9 +/- 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 x 10(-8) M) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.

Androgen-Insensitivity Syndrome↗
PubMed1999

An androgen receptor mutation in the direct vicinity of the proposed C-terminal alpha-helix of the ligand binding domain containing the AF-2 transcriptional activating function core is associated with complete androgen insensitivity.

Subjects with androgen insensitivity syndromes (AIS) are characterized by a 46, XY karyotype, presence of testes, normal or elevated androgen levels in blood, and impairment of the usual response to androgens associated with various aberrations of male differentiation and virilization ranging from slightly undervirilized men to phenotypic females. Here we describe a novel proline to serine mutation in codon 892 (exon 8) of the androgen receptor in a patient with complete androgen insensitivity. The mutation is located in the direct vicinity of the proposed C-terminal alpha-helix of the ligand binding domain containing the AF-2 transcriptional activating function core. Investigation of androgen binding in cultured testicular fibroblasts of the patient revealed a reduced AR binding capacity (11 fmol/mg protein) and a highly elevated Kd value (3.1 nM) in comparison to control genital skin fibroblasts. Cotransfection studies with an androgen-responsive reporter gene revealed a diminished transactivation property of the mutant androgen receptor.

Adult↗
PubMed1999

Complete androgen insensitivity caused by a new frameshift deletion of two base pairs in exon 1 of the human androgen receptor gene.

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5alpha-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.

Adult↗
PubMed1998

Expression of 5alpha-reductase in the human temporal lobe of children and adults.

Androgens exert important biological effects on the brain, and 5alpha-reductase plays a crucial role in androgen metabolism. Therefore, we investigated the expression of the two isozymes of 5alpha-reductase in the human temporal lobe to determine the predominant isoform and to elucidate the existence of possible sex differences and differences between children and adults. We studied biopsy materials from the temporal lobe of 34 women, 32 men, and 12 children. Quantification of 5alpha-reductase 1 and 2 messenger ribonucleic acid (mRNA) was achieved by competitive RT-PCR. 5Alpha-reductase activity was determined in tissue homogenates using [1,2-3H]androstenedione as the substrate. Only 5alpha-reductase 1 mRNA was expressed in human temporal lobe tissue; 5alpha-reductase 2 mRNA was not expressed. 5Alpha-reductase 1 mRNA concentrations did not differ significantly in the cerebral cortex of women [25.9+/-7.9 arbitrary units (aU); mean +/-SEM] and men (20.4+/-2.8 aU) or in the cerebral cortex (23.3+/-4.4 aU) and the subcortical white matter of adults (32.6+/-5.6 aU), but they were significantly higher in the cerebral cortex of adults than in that of children (6.4+/-2.3 aU; P < 0.005). The apparent Km of 5alpha-reduction did not show significant differences between the two sexes. In conclusion, 5alpha-reductase 1 mRNA is expressed in the temporal lobe of children and adults, but 5alpha-reductase 2 mRNA is not. 5Alpha-reductase 1 mRNA concentrations did not differ significantly in the sexes, but they were significantly higher in specimens of adults than in those of children.

Adult↗
PubMed1998

Response to androgen treatment in a patient with partial androgen insensitivity and a mutation in the deoxyribonucleic acid-binding domain of the androgen receptor.

Supplemental androgen therapy has enhanced virilization in only a few patients with partial androgen insensitivity (PAIS). We herein report on virilization in a patient with PAIS and a point mutation in the DNA-binding domain of the androgen receptor. At the age of 19 yr, the patient sought medical attention because of undervirilization. Endocrine findings were typical for androgen insensitivity, but 5alpha-reductase activity and androgen binding characteristics in fibroblasts cultured from genital skin were normal. In an attempt to improve virilization, high dose testosterone enanthate treatment (250 mg by i.m. injection once a week) was begun. After 3.5 yr of this treatment, marked promotion of virilization was achieved, i.e. lowering of voice, male pattern secondary hair distribution, marked growth of beard and coarse body hair, increase in phallic size, increase in bone mineral density, and decrease in mammary gland size. In addition, serum lipid levels were not affected. To our knowledge this is the first documentation of successful treatment in a patient with PAIS and a point mutation in the DNA-binding domain of the androgen receptor.

Adult↗
PubMed1996

Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene.

OBJECTIVE: Androgen insensitivity syndromes (AIS) in subjects with 46, XY karyotype and normal or even elevated androgen blood levels are characterized by various aberrations in male differentiation and virilization. AIS is often accompanied by a broad spectrum of abnormal binding characteristics of the androgen receptor (AR). In order to investigate the correlation between the degree of virilization defect and the type of androgen binding abnormalities and/or the nature of the mutation in the AR gene, we determined androgen binding characteristics of the AR protein and the sequence of the AR gene in clinically and biochemically well characterized patients with various degrees of androgen resistance. DESIGN AND PATIENTS: The activity of 5 alpha-reductase and the binding of androgen to its receptor (KD-values, Bmax, thermolability) were determined in genital skin fibroblasts from 20 patients with various degrees of defects in virilization (2 CAIS, complete AIS; 18 PAIS, partial AIS patients). The AR gene of these 20 subjects was characterized by PCR-SSCP analysis. In case of aberrant electrophoretic mobility the corresponding exon was sequenced. RESULTS: The 2 patients with CAIS and 7 with PAIS showed a mutation in the AR gene. In two, the mutation was in the DNA binding domain, and in all others in the ligand binding domain. In 11 patients with severe virilization defects no abnormal behaviour was detected in the PCR-SSCP. Transcriptional activation studies of two mutant ARs revealed that an approximately tenfold higher androgen concentration (methyltrienolone) is necessary to achieve maximal response as compared to the wild type AR. CONCLUSIONS: There is no obvious relation between the degree of androgen resistance and the binding parameters of the AR and/or the nature of mutation in the AR gene. Androgen insensitivity syndrome can occur despite normal androgen binding and presumably non-mutated AR genes. Even if there is abnormal binding of androgen and/or a mutation in the AR gene there is no clear-cut relationship between these parameters and the degree of virilization defects. Thus, in a proportion of patients, neither the determination of binding parameters of the AR nor the detection of mutations in the AR gene are sufficient to understand the mechanisms underlying the androgen insensitivity syndrome.

Adolescent↗
PubMed1995

Oestrogen formation from androstenedione in human bone.

BACKGROUND AND OBJECTIVE: Peripheral aromatization of testosterone and androstenedione is the principal source for circulating oestrogens in men and in castrated and post-menopausal women. Since human bone is a target organ for androgens and oestrogens, aromatase activity was assessed in human spongiosa obtained from patients who were undergoing orthopaedic surgery. DESIGN AND PATIENTS: In initial experiments for assessing aromatization, oestrogen formation from 1,2,6,7-3H-androstenedione was compared with the release of tritiated water from 1 beta-3H-androstenedione. Since the rates of enzyme activity were similar with the two methods, rates of oestrogen formation were determined under standardized conditions with the tritiated water generation technique in bone specimens obtained from 4 men and 11 post-menopausal women. RESULTS: The apparent Km of the aromatase ranged between 6 and 50 nM (20.4 +/- 3.9; mean +/- SEM), values in the range of those reported for human placental microsomes. The maximum velocity (Vmax) of the aromatase activity ranged between 0.14 and 1.23 nmol/g DNA/h. CONCLUSIONS: Oestrogens formed in human bone may play a physiological role in steroid hormone action in this tissue.

Aged↗
PubMed1994

Decrease in androgen binding and effect of androgen treatment in a case of X-linked bulbospinal neuronopathy.

X-linked recessive bulbospinal neuronopathy is a motoneuron disorder to be distinguished from amyotrophic lateral sclerosis, Effective treatment is not known. Patients with X-linked recessive bulbospinal neuronopathy may show gynecomastia and testicular atrophy, and a mutation in the androgen receptor gene has been found associated with the disease. Intermediate steps leading from the androgen receptor abnormality to the clinical syndrome have not yet been elucidated. Therefore, binding of androgen ([3H]dihydrotestosterone) to its specific receptor by genital skin fibroblasts cultured from a patient with X-linked recessive bulbospinal neuronopathy and confirmed androgen receptor mutation was studied. Markedly decreased binding capacity was found. We treated the patient for 6 months with nandrolone-decanoate. No effect on his neuromuscular status was observed during 2 years of follow-up.

Anabolic Agents↗
PubMed1994

Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias.

OBJECTIVE: Hypospadias is the most common birth defect in males. In most cases the aetiology is unknown. Since penile development is androgen dependent and oestrogen can modify androgen action, we compared the formation of oestrogen in penile tissue from patients with hypospadias to those with normal penile development. DESIGN AND PATIENTS: Oestrogen formation was assessed in fibroblast monolayers grown from biopsies of genital and non-genital skin from 11 males with normal genital development (controls) and 18 males with severe hypospadias utilizing the incorporation of tritium into H2O resulting from the aromatization of 1 beta-3H-androstenedione. RESULTS: In paired fibroblast strains from genital and non-genital skin of nine males with hypospadias, oestrogen formation was significantly (P < 0.025) lower in non-genital skin. Rates of oestrogen formation were also higher in a subset of foreskins from subjects with hypospadias than in normal controls and the remaining hypospadias subjects. In addition, oestrogen formation in this subset of fibroblast strains from patients with hypospadias was markedly enhanced by incubation of intact monolayers with either cholera toxin or forskolin, agents known to stimulate cAMP formation. Oestrogen formation in the remaining cell strains (controls and hypospadias) was also enhanced in most instances by cholera toxin and forskolin, although to a much lower degree. Thus, we identified in the hypospadias group a subgroup of fibroblast strains in which unstimulated and stimulated oestrogen formation was markedly higher than in other strains examined. CONCLUSIONS: Since oestrogen can modify certain androgen effects within cells and since formation of the male genitalia during embryogenesis is mediated by androgens, elevated oestrogen formation in male genital tissue might be a causative factor of hypospadias in some instances.

Adolescent↗
PubMed1994

Juvenile nasopharyngeal fibroma: androgen receptors and their significance for tumor growth.

Since the publications of Martin, et al. (1948) and Schiff (1959), who were the first to report on the administration of sex hormones to juvenile nasopharyngeal fibroma (JNF) patients, several authors have described the different clinical effects and histologic changes after androgen and estrogen application. Since the mechanism of action of sex steroids in juvenile nasopharyngeal fibroma is almost unknown, the authors have studied androgen receptor binding in cultured tumor fibroblasts from three patients with JNF. Maximum androgen binding (Bmax) of the tumor fibroblasts approximated to that of genital skin fibroblasts, which served as a control androgen target tissue with high receptor density. Furthermore, in vitro experiments showed that the growth rate of tumor fibroblasts increased when testosterone was added to the culture medium, while the addition of two antiandrogens, cyproterone and flutamide, caused a reduction in growth rate. It is concluded from these results that JNF is a hormone-dependent tumor stimulated by testosterone whose growth rate may, at least in vitro, be reduced by antiandrogens such as cyproterone and flutamide.

Cell Division↗
PubMed1992

The human androgen receptor: structure/function relationship in normal and pathological situations.

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.

Amino Acid Sequence↗
PubMed1992

Androstenedione metabolism in cultured human osteoblast-like cells.

Bone is a target organ of androgens. The mechanism by which these steroids exert their action within bone cells is still poorly understood. The metabolism of androstenedione, the major circulating androgen in women, was, therefore, assessed in osteoblast-like bone cells cultured from bone of 16 postmenopausal women (mean age, 69 yr; range, 56-80) and 3 elderly men (mean age, 71 yr; range, 69-73) undergoing total hip replacement. Each cell strain was incubated under standardized conditions with varying concentrations of [1,2,6,7-3H]androstenedione (0.05-5 microM). In every instance 5 alpha-reduced metabolites and 17 beta-hydroxysteroids were formed. There was no correlation between the volumetric density of the resected bone and androstenedione metabolism of the corresponding cultured bone cell strains. The apparent Km for the 5 alpha-reductase activity (sum of androstanedione and dihydrotestosterone) of all 19 cell strains was 0.7 +/- 0.1 microM (mean +/- SEM), and the apparent Km for 17 beta-hydroxysteroid dehydrogenase (sum of testosterone and dihydrotestosterone) was 2.3 +/- 0.8 microM (mean +/- SEM), values similar to those reported for other androgen target organs. Our results demonstrate that human osteoblast-like cells have the capacity to transform androstenedione into the more potent biological androgens testosterone and dihydrotestosterone. Since the Km values of both 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase exceed the serum androstenedione concentration, the formation of testosterone and dihydrotestosterone appears to be mainly a function of substrate availability.

Aged↗
PubMed1991

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Amino Acid Sequence↗
PubMed1991

[Hormone dependence of juvenile nasopharyngeal fibroma].

Since Martin (1948) and Schiff (1959) first reported the use of sex hormones in JNF patients, many authors have described the various clinical effects and histological changes found after administration of androgens or estrogens. In 1980, Johns attempted unsuccessfully to detect estrogen receptors in the tissue of tumors from JNF patients. In 1987, however, Farag et al. succeeded in the demonstration of androgen receptors in homogenates of such tumor tissue.--In 1989, the authors were able to determine the uptake and receptor-binding of radioactively labelled dihydrotestosterone in cultured fibroblasts from a tumor from a 16-year-old JNF patient, and to confirm this result in two other cases. The maximum levels of hormone binding to the fibroblasts was much the same as is found with genital skin fibroblasts, included in the study as a control androgenic target-tissue with high receptor-density. At the same time it was demonstrated that it was possible to stimulate the tumor fibroblasts in vitro by adding testosterone to the culture medium. The attempt to block cell growth with the antiandrogen cyproterone acetate, was not successful, however. This can possibly be put down to the high progestogenic activity of this antiandrogen. Further in vitro studies with substances which are purely androgenic (e.g., flutamide) or with the acetate-free form of cyproterone (which has no progestogenic activity) will possibly be of help in the search for a substance capable of blocking tumor growth.

Adolescent↗
PubMed1991

Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics.

We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution (with 15-20% of wild-type androgen-binding capacity), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (with normal androgen-binding capacity, but a rapidly dissociating ligand-receptor complex). The mutations eliminate a Hinfl restriction site. Screening for the loss of the Hinfl site in both families with the Asp----Asn mutation resulted in the recognition of heterozygous carriers in successive generations of each. Both mutant androgen receptors were generated in vitro and transiently expressed in COS and HeLa cells. The receptor proteins produced had the same altered binding characteristics as those measured in fibroblasts from the affected subjects. R1881-activated transcription of a GRE-tk-CAT reporter gene construct was strongly diminished by both mutant receptors and was only partially restored using a 100-fold higher concentration of ligand compared with wild-type receptor. Thus, aspartic acid-686 appears essential for normal androgen receptor function. Substitution of this amino acid residue, by either histidine or asparagine, results in androgen insensitivity and lack of androgen-dependent male sexual differentiation.

Adult↗
PubMed1989

Intracellular and nuclear binding of [3H]dihydrotestosterone in cultured genital skin fibroblasts of patients with severe hypospadias.

Androgens stimulate the development and growth of the male external genitalia. Because hypospadias is the most common congenital defect of the male urethra and because in most cases the cause of this malformation is unknown, we examined the hypothesis that the etiology of the severe forms of this disorder, which is frequently associated with other genital anomalies, might be explained by receptor abnormalities. Intracellular and nuclear binding of androgens were determined in cultured genital skin fibroblasts from 10 males who underwent circumcision for phimosis (controls A), 2 patients with 5 alpha-reductase deficiency (controls B), and 11 patients with severe forms of hypospadias of unknown etiology. Genital skin fibroblast monolayers were incubated for 60 min at 37 degrees C with varying concentrations of [3H]-dihydrotestosterone ([3H]DHT), and specific binding in whole cells and nuclei was measured. Maximum binding (Bmax) in the whole cell assay averaged 0.88 +/- 0.15 fmol . microgram DNA-1 (mean +/- SD) in the control group (controls A, 0.89 +/- 0.16 fmol . microgram DNA-1; controls B, 0.85 fmol . microgram DNA-1) and 0.7 +/- 0.25 fmol . microgram DNA-1 in the patients with hypospadias. In the latter group, Bmax in six patients was below the minimum values determined in the controls. Maximum specific nuclear binding in the control groups averaged 43% (range, 30-55%) of the corresponding intracellular binding. In contrast, nuclear binding in strains from patients with hypospadias was lower (range, 0-12% of whole cell Bmax). In particular, no high affinity saturable nuclear [3H]DHT binding could be measured in 6 of the 11 patients. We interpret these data to suggest that defective intracellular and/or nuclear binding might be the cause of defective genital development in some patients with severe hypospadias.

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