Thioalbamide inhibits FoF1-ATPase in breast cancer cells and reduces tumor proliferation and invasiveness in breast cancer in vivo models.

OBJECTIVE: Thioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target. METHODS: Wild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and "experimental metastasis" mouse models. RESULTS: Experiments carried out on ρ0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the FoF1-ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis" mouse models have highlighted its ability to inhibit breast cancer cell invasiveness. CONCLUSIONS: Overall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment.

Activated-farnesoid X receptor (FXR) expressed in human sperm alters its fertilising ability.

The farnesoid X receptor alpha (FXR) is a bile acid sensor activated by binding to endogenous bile acids including chenodeoxycholic acid (CDCA). Although, FXR is expressed in male reproductive tissue, the relevance of the receptor on reproduction is scarcely known. Here, we demonstrated the FXR presence and its action on several human sperm features. Western blot and immunofluorescence assays evidenced the FXR expression in human spermatozoa and the localisation in the middle piece. CDCA increasing concentrations and GW4064, synthetic ligand of FXR, were used to study the FXR influence on sperm motility, survival, capacitation, acrosome reaction and on glucose as well as lipid metabolism. Interestingly, our data showed that increasing concentrations of CDCA negatively affected sperm parameters, while the receptor blockage by (Z)-Guggulsterone and by the anti-FXR Ab reversed the effects. Intriguingly, elevated CDCA levels increased triglyceride content, while lipase and G6PDH activities were reduced with respect to untreated samples, thus impeding the metabolic reprogramming typical of the capacitated sperm. In conclusion, in this study, we demonstrated for the first time a novel target for FXR and that the activated receptor alters the acquisition of sperm fertilising ability. We showed that sperm itself express the FXR and it is responsive to specific ligands of the receptor; therefore, bile acids influence this cell both in male and in female genital tracts. It might be hypothesized that bile acid levels could be involved in infertility with idiopathic origin as these compounds are not systematically measured in men undergoing medically assisted procreation.

Expression of oestrogen receptors (GPER, ESR1, ESR2) in human ductuli efferentes and proximal epididymis.

Oestrogen targeting in the human genital ducts is still not well-known. In fact, to date, the localization of oestrogen receptors, ESR1 and ESR2, is controversial and the presence of the membrane oestrogen receptor GPER (G protein-coupled oestrogen receptor) is unexplored. This study has investigated the expression of GPER, ESR1, ESR2 in human ductuli efferentes and proximal caput epididymis by immunohistochemistry and Western blot analysis. Furthermore, the presence of PELP1 (proline-glutamic acid-leucine-rich protein 1), a co-regulator of the oestrogen receptors, was also evaluated. In ductuli efferentes, GPER and ESR1 were clearly localized in all epithelial cells, while ESR2 was evidenced only in ciliated cells. Conversely, the epithelial cells of proximal caput epididymis revealed moderate GPER immunoreactivity, the absence of ERS1 and the occasional presence of ESR2. Furthermore, PELP1 was observed in ciliated cells of ductuli efferentes and in principal cells of proximal caput epididymis. Therefore, this study firstly demonstrated the expression of GPER in human male genital ducts, revealing a new mediator of oestrogen action in these anatomical sites. ESR1 and ESR2 were differentially localized in the two genital tracts together with PELP1, but cell sites of ERs and their co-regulator were not homogeneous. So, a different regional/cellular association of GPER with the classical oestrogen receptors was highlighted, suggesting that oestrogen action could be mediated by GPER, ESR1, ESR2 in ductuli efferentes, while by GPER and, occasionally by ESR2, in proximal caput epididymis. This study suggests that the specific oestrogen-mediated functions in human genital ducts might result from the different local interactions of oestrogens with oestrogen receptors and their co-regulators.

Identification of the G protein-coupled estrogen receptor (GPER) in human prostate: expression site of the estrogen receptor in the benign and neoplastic gland.

Estrogens are involved in growth, differentiation and pathogenesis of human prostate through the mediation of the classical estrogen receptors ERα and ERß. The G protein-coupled estrogen receptor (GPER) is a 'novel' mediator of estrogen signaling which has been recently recognized in some human reproductive tissues, but its expression in the prostate gland is still unknown. Here, we investigated GPER in benign (from 5 patients) and neoplastic prostatic tissues (from 50 patients) by immunohistochemical analysis and Western blotting. Normal areas of benign prostates revealed a strong GPER immunoreactivity in the basal epithelial cells while luminal epithelial cells were unreactive and stromal cells were weakly immunostained. GPER was also immunolocalized in adenocarcinoma samples but the immunoreactivity of tumoral areas decreased from Gleason pattern 2 to Gleason pattern 4. Furthermore, a strong GPER immunostaining was also revealed in cells of pre-neoplastic lesions (high-grade prostatic intra-epithelial neoplasia). Western blot analysis of benign and tumor protein extracts showed the presence of a ~42 kDa band, consistent with the GPER molecular weight. An increase in both pAkt and p cAMP-response-binding protein (pCREB) levels was also observed in poorly differentiated PCa samples. Finally, this work identified GPER in the epithelial basal cells of benign human prostate, with a different localization with respect to the classical estrogen receptors. Furthermore, the expression of GPER in prostatic adenocarcinoma cells was also observed but with a modulation of the immunoreactivity according to tumor cell arrangements.

Identification of G protein-coupled estrogen receptor in human and pig spermatozoa.

Estrogens are known to influence functional properties of mammalian spermatozoa inducing rapid responses through the classical estrogen receptors (ERα and ERß). Recently, the G protein-coupled estrogen receptor (GPER) has been identified as mediator of fast non-genomic estrogen effects in different cells. This work investigated the expression of GPER in human and pig spermatozoa using immunofluorescence, Western blot analysis and RT-PCR. GPER was found to be confined to the mid-piece of human sperm cells, whereas it was detected in the acrosomal region, the equatorial segment and the mid-piece of pig spermatozoa. Furthermore, in the male gametes of both species, the immunoblots of sperm extracts revealed a band at ~42 kDa, consistent with the GPER molecular weight, and RT-PCR detected the GPER transcripts. This is the first report demonstrating the expression of GPER in human and pig mature sperm cells and evidencing its species-specific cellular localization.

Insulin and IR-beta in pig spermatozoa: a role of the hormone in the acquisition of fertilizing ability.

Recent studies have revealed that insulin, the main regulator of the glucose homeostasis in somatic cells, is expressed in human spermatozoa which are also able to secrete it. This study investigated the expression of insulin and insulin receptor beta in pig spermatozoa, at immunohistochemical protein and mRNA level. The immunofluorescence assay revealed that insulin and its receptor were co-localized in the sperm midpiece, while insulin was also detected in the acrosomal region. Western blot evidenced a 36 kDa band for insulin and a 95 kDa band for insulin receptor, such as reported in somatic cells. In addition, both insulin and insulin receptor transcripts were detected in pig spermatozoa. Interestingly, a possible biological role of the hormone was evidenced during pig sperm capacitation and acrosome reaction. In fact, the results showed that insulin (0.01 and 0.1 nm) can induce both the activities. A possible autocrine short loop of insulin in pig spermatozoa was suggested by the evaluation of the hormone secretion in both uncapacitated and capacitated spermatozoa. Furthermore, spontaneous sperm capacitation and acrosome reaction were stimulated by glucose and inhibited by the blockage of insulin release (nifedipine). In conclusion, this work has firstly demonstrated the expression of insulin and of its receptor, as well as the insulin secretion by pig spermatozoa, thereby suggesting an unexpected significance of the hormone in the acquisition of the male gamete fertilizing ability.

Evidence that bergapten, independently of its photoactivation, enhances p53 gene expression and induces apoptosis in human breast cancer cells.

Psoralens (5-MOP and 8-MOP), a class of naturally occurring compounds, in combination with ultraviolect light are potent modulators of epidermal cell growth and differentiation. For a long time, photo-chemotherapy has been used in the treatment of psoriasis where it can reduce the number of cycling keratinocytes and decrease the IGF-1 receptors. However, the molecular mechanism of PUVA therapy remains unclear. In this study, we have evaluated, for the first time, in MCF-7 and SKBR-3 breast cancer cells the effects of 5-MOP (Bergapten), independently of its photoactivation, on the signalling pathways involved in cell cycle arrest and in apoptosis. Drug treatment induced a block in the G0/G1 phase and increased mRNA and protein levels of p53 and p21waf. These data correlate with a functional activation of caspase 8/caspase 9 together with DAPI staining and DNA ladder. Bergapten can transactivate p53 gene promoter in these cells and site-direct mutagenesis studies showed that the binding sequence of the nuclear factor NF-Y on p53 promoter is required for 5-MOP responsiveness. Besides, Bergapten increases NF-Y nuclear translocation through p38 MAPK activation. The same treatment impairs the PI3Kinase/AKT survival signal, in hormone-dependent MCF-7 cells even in the presence of IGF-I/E2 mitogenic factors. Here, we demonstrated that Bergapten, independently on the exposure to UV, generates membrane signalling pathways able to address apoptotic responses in breast cancer cells and to counteract the stimulatory effect of IGF-I/E2 on estrogen-receptor positive MCF-7 cell growth and progression.

The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells.

Previous epidemiological reports have suggested that red wine intake is associated with beneficial health effects due to the ability of certain phytochemical components to exert estrogen-like activity. It has been also documented that estrogens induce the proliferation of hormone-dependent breast cancer cells by binding to and transactivating estrogen receptor (ER) alpha, which in turn interacts with responsive DNA sequences located within the promoter region of target genes. In order to provide further insight into the positive association between wine consumption and the incidence of breast carcinoma in postmenopausal women, we have evaluated the estrogenic properties of two abundant wine-derived compounds, named piceatannol (PIC) and myricetin (MYR), using as model systems the hormone-sensitive MCF7 and the endocrine-independent SKBR3 breast cancer cells. On the basis of our experimental evidence PIC and MYR may contribute to the estrogenicity of red wine since: (1) they transactivate endogenous ER alpha; (2) they activate the agonist-dependent activation function (AF) 2 of ER alpha and ER beta in the context of the Gal4 chimeric proteins; (3) they rapidly induce the nuclear immunodetection of ER alpha; (4) they regulate the expression of diverse estrogen target genes; (5) they compete with 17beta-estradiol for binding to ER alpha and ER beta; and--as a biological counterpart of the aforementioned abilities--(6) they exert stimulatory effects on the proliferation of MCF7 cells. Hence, the estrogenic activity of PIC and MYR might be considered at least as a potential factor in the association of red wine intake and breast tumors, particularly in postmenopausal women.

Antioxidant capacity in seminal plasma of transfusion-dependent beta-thalassemic patients.

BACKGROUND: Homozygous beta-thalassemia is a haemolytic disorder with a high potential for oxidative damage, due to the high circulating iron levels. Enzymatic and non enzymatic antioxidant capacities, as well as lipoperoxide content, were investigated in seminal plasma of these patients to evaluate a possible oxidative stress. METHODS: Semen samples from 10 transfusion-dependent beta-thalassemic patients and 18 control subjects were examined. The assessment of the seminal antioxidant capacity included spectrophotometrical assays for determination of superoxide dismutase and catalase activity and of the total antioxidant status value. Furthermore, malondialdehyde level was detected as marker of lipoperoxidation. RESULTS: All the beta-thalassemic patients showed high serum ferritin levels, progressive sperm motility below 50 %, and normal sperm count (median: 43 x 10 (6) sperm/ml). Increased superoxide dismutase ( p < 0.01) and catalase ( p < 0.001) activities, but unaltered total antioxidant status values, were detected in the patients' seminal plasma. Furthermore, augmented malondialdehyde levels ( p < 0.001) were measured in the patients. CONCLUSIONS: Seminal antioxidant pattern of iron overloaded beta-thalassemic patients indicated the hyperactivation of the enzymatic free-radical scavengers which could be explained as a compensatory mechanism to possible high levels of reactive oxygen species. Furthermore, the increase of seminal lipoperoxidation suggested an oxidative stress in semen of these patients and it could have contributed to the impairment of sperm motility.

Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs.

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.

Aromatase immunolocalization in human ductuli efferentes and proximal ductus epididymis.

Abstract Cytochrome P450 aromatase is a terminal enzyme that catalyses the conversion of androgens into oestrogens. This study investigated the immunohistochemical localization of aromatase in human efferent ductules and proximal ductus epididymis using a mouse anti-human monoclonal P450arom IgG as primary antibody and a goat anti-mouse biotinylated IgG as secondary antibody. A strong immunoreaction was observed in the epithelial cell cytoplasm of both ductuli efferentes and proximal ductus epididymis, whereas the smooth muscle cells were immunonegative in the two regions. The results show, for the first time in humans, that epithelial cells of ductuli efferentes and proximal caput epididymis express aromatase, suggesting that locally produced oestrogens may have a role in epididymal function.

Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha.

Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ERbeta and the hormone-binding domains of both ERalpha and ERbeta, BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.

Breast cancer: from estrogen to androgen receptor.

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

The direct proliferative stimulus of dehydroepiandrosterone on MCF7 breast cancer cells is potentiated by overexpression of aromatase.

In women after menopause aromatization of adrenal androgens represents the main source of estrogens, which may promote the development of hormone-dependent breast tumor. Several studies have attempted to determine the cell type within carcinomas that is responsible for 'in situ' estrogen biosynthesis and whether the amount produced may sustain relevant biological effects. Here we show P450arom mRNA and protein expression together with immunocytochemical localization of aromatase in the epithelial MCF7 breast cancer cell line. Moreover, we demonstrate that the enhanced aromatization of dehydroepiandrosterone in aromatase transfected MCF7 cells confers biological advantages such as proliferative stimulation similar to that induced by estradiol. Our results suggest that aromatase inhibitors may be particularly effective in the treatment of hormone-dependent breast cancer disease in postmenopausal women.

Impaired seminal antioxidant capacity in human semen with hyperviscosity or oligoasthenozoospermia.

Antioxidant capacity of seminal plasma was evaluated in 120 semen samples subdivided into asthenozoospermic and oligoasthenozoospermic specimens with normal consistency and into asthenozoospermic and oligoasthenozoospermic specimens with hyperviscosity. Semen samples (n = 25) from normozoospermic donors were used as a control group. Scavenger antioxidant capacity of reactive oxygen species was evaluated by superoxide dismutase and catalase activity measurements, whereas the chain-breaking antioxidant efficiency was detected by total antioxidant status assessment. In semen with normal viscosity, unaltered enzymatic and nonenzymatic antioxidant capacity was revealed in the asthenozoospermic specimens, whereas low superoxide dismutase activity was detected in oligoasthenozoospermic samples. On the contrary, impairment of both the scavenger and chain-breaking antioxidative systems was revealed in asthenozoospermic and oligoasthenozoospermic hyperviscous ejaculates, regardless of sperm count. Catalase activity and total antioxidant status values were also reduced in the 2 subgroups of hyperviscous ejaculates compared with their respective matched controls, whereas similar superoxide dismutase activities were detected in oligoasthenozoospermic samples with normal and high consistencies. These results suggest that asthenozoospermia could be related to an antioxidant deficiency only in combined ejaculate pathologies, and that a severe impairment of the low and high molecular weight seminal antioxidative capacities could be associated with semen hyperviscosity.

Immunolocalization of cytochrome P450 aromatase in rat testis during postnatal development.

Aromatization of androgens into estrogens in rat testis is catalyzed by the microsomal enzyme cytochrome P450 aromatase. In this work, aromatase cellular site was investigated in prepuberal, peripuberal and postpuberal testis, from 10-, 21- and 60-day-old rats respectively. Paraffin-embedded testis sections were processed for P450arom immunostaining using a rabbit polyclonal antiserum generated against purified human placental cytochrome P450 aromatase. Next, biotinylated anti-rabbit IgG was applied, followed by ABC/HRP/complex amplification with diaminobenzidine as chromogen. Prepuberal testis sections showed a strong immunoreactivity of aromatase in Sertoli cell cytoplasm while interstitial cells were immunonegative. In peripuberal testis sections, cytoplasmic immunoreaction was weak in Sertoli cells, but it was strong in spermatocytes and sporadic in Leydig cells. Postpuberal testis sections displayed a moderate aromatase immunoexpression in spermatocytes while a strong immunostaining was observed in round and elongated spermatids, as well as in Leydig cells. These results indicate a different age-dependence of aromatase localization in rat testicular cells during gonadal development. In particular, inside the seminiferous tubules, the aromatization site moves from Sertoli cells to late germ cells, suggesting a proliferative role of aromatase in prepuberal testis and its subsequent involvement in meiotic and post-meiotic germ cell maturation.

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells.

Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T(3)) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T(3) on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N(6),2'-O-dibutyryladenosine-3':5'-cyclic monophosphate ((Bu)(2)cAMP) that was clearly down-regulated by T(3). The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [(3)H]H(2)O released after incubation with [1 beta-(3)H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)(2)cAMP and markedly down-regulated by T(3). In contrast, the strong increase in aromatase mRNA upon (Bu)(2)cAMP stimulation was apparently unaffected by T(3) administration. This paper shows how the identification of an altered transcript induced by T(3) coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T(3)-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T(3) on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T(3) in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.

Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone.

Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3.

Prostatic origin of a zinc binding high molecular weight protein complex in human seminal plasma.

The profile of the zinc ligand high molecular weight proteins was investigated in the seminal plasma of 55 normozoospermic subjects by size exclusion high performance liquid chromatography (HPLC). The proteins were recovered from Sephadex G-75 gel filtration of seminal plasma in three zinc-containing fractions which were then submitted to HPLC analysis. The results were, that in all the samples, the protein profiles showed two peaks with apparent molecular weight of approximately 660 and approximately 250 kDa. Dialysis experiments revealed that both approximately 660 and approximately 250 kDa proteins were able to uptake zinc against gradient indicating their zinc binding capacity. The HPLC analysis of the whole seminal plasma evidenced only the approximately 660 kDa protein complex as a single well quantifying peak, furthermore a positive correlation between its peak area and the seminal zinc values (P < 0.001) was observed. This suggested a prostatic origin of the approximately 660 kDa protein complex which was then confirmed by the seminal plasma HPLC analysis of a subject with agenesis of the Wolffian ducts. Finally the study demonstrated the presence of two zinc binding proteins, approximately 660 and approximately 250 kDa respectively, in human seminal plasma and the prostatic origin of the approximately 660 kDa.

[Thesis for induction into the Venezuelan Society for the History of Medicine: Medical deontology in Venezuela]. / Trabajo de incorporación, como Individuo de Número, a la Sociedad Venezolana de Historia de la Medicina. Historia de la Deontologia Medica en Venezuela.

After a brief reference to his predecessor, the author presents a review on deontological principles put to use in the Venezuelan medicine, from the discovery to this century, after some considerations on the roles played by the state, the universities and the society. Finally, the importance of the moral compromise of the medical doctor as a professional who must have a faultless behaviour is highlighted.