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A Poletti

Publications and source records attributed to A Poletti.

At least 37 records · Page 2Linked to original sources
PubMed1998

Presence of 5alpha-reductase isozymes and aromatase in human prostate cancer cells and in benign prostate hyperplastic tissue.

BACKGROUND: Prostate trophism depends on DHT formed from T by the enzyme 5alpha-R. Two 5alpha-R isoforms with different biochemical characteristics have been cloned. Also estrogens might contribute to the prostate growth; however, their intraglandular formation by the enzyme aromatase is still debated. The aim of the present study was to verify whether (a) only one or both isoforms of the 5alpha-Rs are expressed in the prostate cancer cell line LNCaP and in BPH, or (b) the aromatase is present in these samples. METHODS: The profile of the pH optimum of the 5alpha-Rs was evaluated "in vitro" in LNCaP cells by the production of labeled 5alpha-reduced metabolites either from [14C]-T or [14C]-D4 at pH 3.5-8. The gene expression of the two 5alpha-Rs and of the aromatase in LNCaP cells and in BPH specimens was analyzed by RT-PCR combined to Southern blot analysis, using specific sets of oligonucleotides. The tissue localization of 5alpha-R1 was analyzed by immunohistochemistry using an anti-5alpha-R1 polyclonal antibody. RESULTS: (a) In LNCaP cells, the formation of 5alpha-reduced metabolites from the respective precursors increases progressively as a function of pH, being the highest at neutral pH values; (b) the 5alpha-R1 isoform is expressed in both LNCaP cells and in BPH, while the 5alpha-R2 mRNA is present only in BPH, but not in LNCaP cells; and (c) no aromatase transcripts were observed either in BPH or in LNCaP cells. CONCLUSIONS: A careful examination of the possible differential expression of T-activating enzymes, particularly in prostate cancer, would be of help to choose the appropriate treatment.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

The 5alpha-reductase in the central nervous system: expression and modes of control.

The present paper will summarize two important aspects of the interactions between steroids and the brain, which have recently been studied in the authors' laboratory. In particular the paper will consider data on: (1) the significance of the two isoforms of the 5alpha-R during brain ontogenesis and development, and (2) the cross-talk between glial and neuronal elements, particularly in relation to the metabolism of sex hormones. (1) The data obtained have shown that the 5alpha-R type 1 enzyme is constitutively expressed in the rat CNS at all stages of brain development. Moreover, the expression of the 5alpha-R type 1 is similar in males and in females, and does not appear to be controlled by androgens. The gene expression of the 5alpha-R type 2 is totally different. This isoform appears to be expressed in the rat brain almost exclusively in the late fetal/early post-natal life and is controlled by testosterone. (2) The present data show that two cell lines derived respectively from a rat glioma (C6 cell line) and from a human astrocytoma (1321N1 cell line) are able to convert testosterone and progesterone into their corresponding 5alpha-reduced metabolites dihydrotestosterone and dihydroprogesterone. The possibility that secretory products of normal and tumoral brain cells might be able to influence steroid metabolism occurring in the two glial cell lines previously mentioned has been considered.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

Transient expression of the 5alpha-reductase type 2 isozyme in the rat brain in late fetal and early postnatal life.

The enzyme 5alpha-reductase plays a key role on several brain functions controlling the formation of anxiolytic/anesthetic steroids derived from progesterone and deoxycorticosterone, the conversion of testosterone to dihydrotestosterone, and the removal of excess of potentially neurotoxic steroids. Two 5alpha-reductase isoforms have been cloned: 5alpha-reductase type 1 is widely distributed in the body, and 5alpha-reductase type 2 is confined to androgen-dependent structures. In this study, the gene expression of the two 5alpha-reductase isozymes has been analyzed in fetal, postnatal, and adult rat brains by RT-PCR followed by Southern analysis. 5Alpha-reductase type 1 messenger RNA is always detectable in the rat brain [from gestational day 14 (GD14) to adulthood]. 5Alpha-reductase type 2 messenger RNA expression is undetectable on GD14, increases after GD18, peaks on postnatal day 2, then decreases gradually, becoming low in adulthood. This pattern of expression appears to be correlated with the rate of production of testosterone by the testis. The possible control by androgens of gene expression of the two isozymes has been studied in brain tissues of animals exposed in utero to the androgen antagonist flutamide; the sex of the animals was determined by genetic sex screening of the SRY gene located on the Y-chromosome. In the brain of male embryos, flutamide treatment inhibited the expression of 5alpha-reductase type 2; this effect was much less pronounced in females. Moreover, 5alpha-reductase type 2 gene expression in cultured hypothalamic neurons is highly induced by testosterone and by the phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate. The transient, androgen-regulated, expression of 5alpha-reductase type 2 overlaps the critical period of development, which may be important for sexual differentiation of the brain and for the formation of anxiolytic/anesthetic steroids involved in the stress responses associated with parturition.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1998

Detection of B-cell monoclonality in fine needle aspiration by PCR analysis.

Cytologic examination of fine-needle aspiration (FNA) sometimes fails to diagnose the malignant nature of B-cell proliferations. In this study we analyzed the Ig gene rearrangement of 49 FNA samples by polymerase chain reaction (PCR) in order to evaluate whether molecular analyses can improve the accuracy of FNA. Twenty-six patients had non-Hodgkin's lymphoma, 11 had reactive lymphoid diseases, 5 had chronic inflammation and 7 had carcinoma. A semi-nested PCR was performed using an oligoprimer specific for consensus sequences of the V regions (FR3A) and two oligoprimers derived from conserved sequences of the J regions (LJH and VLJH). Histologic examination always followed the molecular and cytologic analysis. The sensitivity of PCR and FNA morphological examination in detecting a neoplastic pattern was 92% and 78%, respectively. When samples were considered inadequate for cytologic examination, PCR always reached a diagnosis consistent with the histologic features. Our results demonstrate that PCR analysis of FNA specimens is a reliable and sensitive method capable of enhancing the diagnostic accuracy of cytologic examination.

B-Lymphocytes↗
PubMed1997

Identification of type 1 5alpha-reductase in myelin membranes of male and female rat brain.

The formation of the 5alpha-reduced metabolites of testosterone (T) and of progesterone (P) is a very active process in the brain, since the enzyme 5alpha-reductase (5alpha-R) is present in almost any central nervous system (CNS) structure. A particularly elevated 5alpha-R activity has been shown in myelin sheaths. Two isoforms of the enzyme have been cloned, with different localisation as well as different biochemical properties. The present study was performed to determine whether both isoforms of the 5alpha-R, or only one of them, are/is responsible for the enzymatic activity observed in myelin. Kinetic analyses have been performed on purified myelin membranes prepared from the male or female rat brain, using both T and P as substrates. The 5alpha-R present appears to possess a pH optimum at basic values. The Vmax values obtained in the Lineweaver-Burk analysis were comparable in male and female preparations independently on whether T or P were used as the substrates, suggesting that a single enzymatic form is present in all samples examined; the Km obtained using [14C]T (Km: male 1.14 microM; female 1.46 microM) or [14C]P (Km: male 0.5 microM; female 0.64 microM) as substrates, were in good agreement with those obtained for the recombinant type 1 isoform. These data suggest that the type 1 isoform is the most relevant 5alpha-R present in myelin. To confirm this, a new polyclonal antibody was raised against the type 1 5alpha-R enzymatic protein, and used in immunohistochemical studies. The experiments were performed on the optic nerve, a myelinated structure very rich in 5alpha-R activity and the results clearly indicated the presence of a specific type 1 enzyme immunoreactivity in the myelin sheaths of axons.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1997

Epstein-Barr virus-associated post-transplant lympho-proliferative disease of donor origin in liver transplant recipients.

BACKGROUND/AIMS: Post-transplant lymphoproliferative disease, a potential complication of solid organ transplantation, occurs in about 3% of orthotopic liver transplant recipients. We report the genetic and virological characterization of two cases of post-transplant lymphoproliferative disease that occurred early (4 and 6 months) after orthotopic liver transplant as large-cell non-Hodgkin's lymphomas located at the hepatic hilum. METHODS: Lymphomatous tissues were analyzed for clonality and presence of Epstein-Barr virus (EBV) sequences by Southern blot, polymerase chain reaction, and in situ hybridization techniques. RESULTS: The tumors in both cases were sustained by a clonal proliferation of B lymphocytes containing type A EBV DNA. Moreover, in situ hybridization with a digoxigenin-labeled EBV-specific probe evidenced a strong nuclear signal in most of the neoplastic cells. DNA microsatellite analysis at three different loci detected alleles of donor origin in both tumor samples, suggesting that the neoplastic B cells were of donor origin. CONCLUSIONS: EBV-infected donor B lymphocytes might be responsible for intragraft post-transplant lymphoproliferative disease in orthotopic liver transplant recipients. As 20 to 30% of post-transplant lymphomas involve the graft itself, donor-derived post-transplant lymphoproliferative disease might be more frequent than presently appreciated. Prospective studies are needed to assess its real incidence and identify possible risk factors.

B-Lymphocytes↗
PubMed1997

Steroid metabolism in the mammalian brain: 5alpha-reduction and aromatization.

Several steroid molecules, including androgens, estrogens, progestagens, and corticostereroids, are able to modulate the brain development and functions. These compounds are not always active in their own natural molecular configuration but they often need to be transformed at the level of their target cells into 'active metabolites'. The two major metabolic pathways that transform steroids in the brain are: the 5alpha-reductase-3alpha-hydroxy-steroid dehydrogenase and the aromatase pathways. Both are present in the brain and probably exert specific roles in the mechanism of action of hormonal steroids. In this article we briefly review some important findings achieved in our own and in other laboratories concerning the cellular and subcellular brain distribution, development, regulation, cloning, and molecular characterization of the involved enzymes. In particular, the recent identification of two isoforms of the 5alpha-reductase, the type 1 and type 2, possessing different structural, biochemical, and distribution characteristics has attracted a considerable attention. The few data available on their brain distribution have been carefully considered. Finally, we have tried to focus on the role of the steroid metabolites in the brain, both when they interact with genomic and with membrane receptors. In particular, some unpublished observations on the effects of two 5alpha-reductase inhibitors on progesterone-induced anesthesia, a phenomenon mediated through the GABA(A) receptor, are presented.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Surgical pathology of primary cardiac and pericardial tumors.

OBJECTIVE: Retrospective study of surgical pathology experience on cardiac and pericardial tumors at the University of Padua in the era of immunohistochemistry and endomyocardial biopsy. METHODS: In the period 1970-1995, we studied 125 bioptic primary neoplasms: specimens were obtained from surgical resection in 116 cases, heart transplantation in 3, pericardiectomy in 3, endomyocardial biopsy in 2 and thoracoscopic biopsy in 1. Tumor histotype was established by light microscopy and more recently by immuno-histochemistry, using a large panel of antibodies, through avidin-biotin peroxidase method, against factor VIII-related antigen, ulex-europaeus, desmin, myoglobin, muscle-specific actin, smooth muscle-specific actin, vimentin, cytokeratins, leukocytic common antigen, neurofilaments and S100-protein. RESULTS: One hundred and thirteen were benign neoplasms: myxoma was the most frequent (87 cases) followed by pericardial cyst (8), endocardial papilloma (5), fibroma (3), rhabdomyoma (3), hematic cyst (2), teratoma (2), hemangioma (1), celothelioma (1) and lipoma (1). Malignancy was diagnosed in 12 cases, and consisted of pericardial mesothelioma (3), myxosarcoma (3), angiosarcoma (2), fibrosarcoma (2) and leiomyosarcoma (2); 4 of them were intracavitary atrial masses and were supposed to be atrial myxoma on the clinical ground. Differential diagnosis included endocardial thrombosis (10), metastasis of concealed extracardiac tumors (5), echinococcosis (3), and Loeffler's fibroplastic endocarditis (3). In 4 cases, cardiac mass histotype was defined without thoracotomy, through endomyocardial (3) and thoracoscopic (1) biopsy. CONCLUSIONS: A large spectrum of cardiac tumors is observed in the surgical pathology practice. Although the diagnosis of cardiac masses is easily attainable by routine imaging techniques, differential diagnosis between primary and secondary tumors, malignant and benign forms, and non neoplastic masses, is achievable only by a thorough microscopic study of surgical resections. The role of the cardiac pathologist is becoming crucial as in other fields of oncology.

Adolescent↗
PubMed1997

Expression of androgen-activating enzymes in cultured cells of developing rat brain.

Dihydrotestosterone and estradiol, two active metabolites formed locally in the brain from testosterone, modulate several functions of the developing rat CNS; these compounds derive from the 5 alpha-reduction or the aromatization of the A-ring of the hormone. Also, progesterone and corticosteroids may be 5 alpha-reduced and subsequently 3 alpha-hydroxylated, becoming modulators of specific neuronal functions. Although the aromatase is a single enzyme, two types of 5 alpha-reductase have been cloned, showing peculiar biochemical properties and probably different functions. Therefore, the isoform(s) of the enzyme 5 alpha-reductase(s) present in early stage of brain development have been characterized in primary neuronal and glial cell cultures obtained from the fetal or neonatal rat brain, respectively. Aromatase expression was also studied. The results have shown that in all the brain cells examined type 1 5 alpha-reductase mRNA is expressed. No specific transcript of type 2 5 alpha-reductase is detectable in any of the cell types examined. Finally, the aromatase gene is expressed only in cultured fetal neurons and especially in those derived from the hypothalamic area of the rat embryos. It is interesting that no aromatase mRNA is detectable in mixed glia or in type 1 astrocytes and oligodendrocytes cultured separately.

Aging↗
PubMed1997

Epidemiology of dilated cardiomyopathy. A prospective post-mortem study of 5252 necropsies. The Heart Muscle Disease Study Group.

UNLABELLED: Dilated cardiomyopathy is a heart muscle disease of unknown aetiology, characterized by left ventricular dilatation and impaired systolic function. Data on the incidence and prevalence of the disease is ambiguous, due to geographic variations, patient selection and the diagnostic criteria adopted. METHODS: All the post-mortem and clinical cases observed in a consecutive series of 5252 patients resident in Trieste during the period November 1987-November 1989 were studied. RESULTS: Incidence of the disease discovered at autopsy was estimated at 4.5/100,000/year (24 cases), while clinical incidence in the same period was 2.45/100,000/year (13 cases). This is a total incidence of 6.95/100,000 new cases a year. A possible family history of heart muscle disease was found in three patients (12.5%). In 15 patients (62.5%) deaths were due to cardiological complications. Endocardial thickening (P = 0.03), fatty infiltration (P = 0.01) and arterial involvement (P = 0.04) were found more frequently in older patients (> 65 years). CONCLUSIONS: The study confirms that dilated cardiomyopathy in Europe has a higher incidence than previously suggested and emphasizes the need for greater diagnostic sensitivity, particularly since pharmacological treatment is now so effective.

Adult↗
PubMed1997

Expression of the protooncogene jun is induced in the rat pancreas by cerulein infusion.

Cholecystokinin (CCK) can stimulate secretion and DNA synthesis in pancreatic acinar cells. Hyperstimulation with cerulein (a CCK analogue) induces acute edematous pancreatitis. To study the effects of in vivo pancreatic stimulation with cerulein, we analyzed the expression of the protooncogenes jun, myc, and fos on the mRNA and protein levels. RNA and protein were extracted from the pancreas of rats administered an infusion of cerulein, 10 micrograms/kg/h (Group A) or 0.25 microgram/kg/h (Group B), or saline (Group C) and sacrificed 2, 4, and 6 h after beginning the infusion and 0, 12, and 24 h and 2, 4, and 6 days after completing the infusion period. Transcript levels were studied using slot-blot analysis. Protein expression was studied using Western blot and immunohistochemistry. No changes were found for the expression of protooncogenes myc and fos on either the transcript or the protein levels. Higher jun mRNA levels were found in Group A than in Group B or C, particularly after 2 h of infusion and 12, 24, and 48 h after the end of a 12-h cerulein infusion. No significant difference was observed in Groups B and C. The jun protein behavior was similar in Groups A and B, revealing two peaks: one early during infusion and a second one after the end of a 12-h cerulein infusion. Jun protein was found mainly in the acinar cells. In conclusion, (1) acinar cells in the rat pancreas respond to cerulein stimulation by increasing the expression of jun; (2) in vivo high doses of cerulein increase the jun mRNA and jun protein levels, whereas low doses raise only the protein levels; and (3) myc and fos are apparently uninfluenced by cerulein administration.

Animals↗
PubMed1997

Phosphorylation of human progesterone receptor by cyclin-dependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo.

The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.

Amino Acid Sequence↗
PubMed1997

Remission in hairy cell leukemia-variant following splenic radiotherapy alone.

The therapeutic approach to hairy cell leukemia (HCL) is in some instances still debated. A variant form of HCL (HCL-V) characterized by high white cell count, splenomegaly, hypercellular and aspirable bone marrow has been described; differential diagnosis often arises with some other B-cell disorders which also show circulating hairy or villous lymphocytes. Conventional treatment for HCL is often less effective in HCL-V. In this report we describe a case with features consistent with HCL-variant treated with splenic radiotherapy. We not only obtained an hematological response but also the near total disappearance of bone marrow infiltration, compatible with a clinical complete remission. Clinical and biological implications of this phenomenon are discussed on the basis of this unexpected therapeutic result, obtained with splenic radiotherapy alone.

Aged↗
PubMed1996

Characterization of rat 5alpha-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae.

Two isoforms of the rat 5alpha-reductase (5alpha-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other delta4-3-keto steroids (e.g. progesterone and corticoids) into their 5alpha-reduced metabolites, have been cloned. In this study, a convenient and efficient system was developed to overexpress the two isoenzymes in Saccharomyces cerevisiae by using the ubiquitin-fusion expression system. Two yeast expression vectors have been prepared, YEpR1 and YEpR2, which code for 5alpha-R type 1 and 5alpha-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5alpha-R type 1 or 5alpha-R type 2 cDNAs in frame to the 3' end of the ubiquitin cDNA. The activity of the two isoenzymes produced in yeast was determined in cell lysates at the enzyme pH optima (type 1, pH 7.5; type 2, pH 5.5) and a possible differential intracellular distribution was also evaluated. The kinetic parameters were: type 1, Km 4.6 microM, Vmax.100.6 micrograms/h per mg of protein; type 2, Km 68.6 nM, Vmax. 0.84 micrograms/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5alpha-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5alpha-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000g). The data indicate that yeasts overexpress the two 5alpha-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell.

Animals↗
PubMed1996

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII.

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.

Adolescent↗