Biomedical subjects

S Minucci

Publications and source records attributed to S Minucci.

At least 73 records · Page 4Linked to original sources

PubMed1996

RAR and RXR selective ligands cooperatively induce apoptosis and neuronal differentiation in P19 embryonal carcinoma cells.

Retinoids cause differentiation in embryonal carcinoma (EC) cells, thus mimicking events in mammalian development. Here, we show that retinoids also cause apoptosis in P19 EC cells. Characteristic DNA fragmentation was observed within 36 h after addition of retinoic acid (RA). Synthetic retinoids that are selective for RA receptors (RAR) were also effective in inducing apoptosis, whereas RXR selective ligands were without effect. The combination of RAR and RXR ligands resulted in a synergistic increase in apoptotic cell death. As with apoptosis, neuronal differentiation of P19 cells was synergistically induced by the combination of RAR and RXR ligands. Data obtained with an RAR antagonist and with P19 cells carrying a dominant negative RXR indicate that the two processes are receptor mediated. Together, our results indicate that retinoid-induced apoptosis and neuronal differentiation are closely coupled, and that both RAR and RXR play a role in these processes as active receptors for their respective ligands.

Apoptosis↗

PubMed1996

Molecular cloning of a novel mRNA (harderin) specifically and highly expressed in the Harderian gland of the frog, Rana esculenta.

A cDNA clone encoding a novel mRNA (harderin) has been isolated from the cDNA library of frog Rana esculenta Harderian gland. The cDNA is 875 bp long and encodes a novel protein of 218 amino acid residues. The deduced proteins reveals no significant homology to other proteins. The mRNA is a single 0.9 kb transcript detected only in the RNA of frog Harderian gland, highly expressed throughout the year. The constant and exclusive presence of harderin in the frog Harderian gland, suggests a constitutive tissue specific expression for this protein.

Amino Acid Sequence↗

PubMed1995

Efficient inhibition of activation-induced Fas ligand up-regulation and T cell apoptosis by retinoids requires occupancy of both retinoid X receptors and retinoic acid receptors.

Two retinoic acid (RA) receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been identified. All-trans-RA and its 9-cis-isomer are ligands for RARs, but only 9-cis-RA binds RXRs with high affinity. Activation-induced T cell hybridoma death is mediated via the engagement of Fas by activation-up-regulated Fas ligand, and RA prevents this type of apoptosis by inhibiting the induction of Fas ligand expression. To investigate the mechanism of RA action, T hybridoma cells were transfected with cDNA encoding RXR beta or dominant-negative RXR beta. Cells that overexpressed RXR beta were more sensitive to 9-cis-RA rescue from activation-induced death than cells transfected with vector alone. In contrast, cells expressing the dominant-negative RXR beta could not be rescued from death with 9-cis-RA. In wild type cells, an RAR-selective synthetic retinoid had little effect on activation-induced apoptosis, while an RXR-selective agonist prevented apoptosis but only at concentrations about approximately 10-fold greater than that required for 9-cis-RA. Simultaneous addition of the RAR- and RXR-selective retinoids completely prevented activation-induced apoptosis at concentrations where either alone had relatively little protective effect. The same hierarchy of efficacy was found for activation-induced Fas ligand expression. These data demonstrate that binding of both RARs and RXRs is required for efficient inhibition of activation-induced Fas ligand upregulation and T cell apoptosis by retinoic acid.

Animals↗

PubMed1995

Detection of c-mos related products in the dogfish (Scyliorhinus canicula) testis.

The objective of the present paper was to do a comparative study to assess somatic versus germ cell localization of c-mos products in the testis. In mouse and amphibian oocytes, c-mos activity is necessary for meiotic maturation. Lack of c-mos expression has been reported in somatic cells of male and female gonads while transcripts have been found in germ cells of testis and ovary. Using a v-mos probe, we report here the detection of a c-mos related transcript (1.7 kb) in the dogfish Scyliorhinus canicula testis. Western blot analysis detects two proteins of 106 and 32 kDa. A specific immunostaining was exclusively localized in the interstitial tissue while the germinal compartment was completely negative. In conclusion, our results indicate for the first time the presence of c-mos products in an elasmobranch species and, moreover, their presence in somatic testicular cells rather than germ cells. Therefore, this finding in an ancient vertebrate indicates that c-mos activity does not have a direct universal role in the regulation of spermatogenesis.

Animals↗

PubMed1995

Ethane 1,2-dimethane sulfonate effects on the testis of the lizard, Podarcis s. sicula Raf: morphological and hormonal changes.

Ethane 1,2-dimethane sulfonate (EDS) destroys Leydig cells in the testis of some rodents (mice excluded), disrupts interstitial and germinal compartments in the frog, Rana esculenta, while it stimulates testicular activity in the teleost, Gobius paganellus. In the Japanese quail the toxin removes mature spermatozoa. There is no information on EDS effects in reptiles. The present study examines the effect of EDS treatment in the lizard Podarcis s. sicula Raf during two different periods of the testicular cycle (winter stasis and breeding season). Animals received a single EDS injection (100 mg/kg body wt) and were sacrificed at 0 and 24 hr and 3, 5, 7, 11, and 28 days after injection. Androgens were measured in plasma and right testes, while left testes were examined histologically. Plasma androgen levels decreased 5-7 days after EDS injection, alongside interstitial tissue destruction and mast cell appearance, with slight but significant increases on Days 11 and 28. Testicular androgen levels did not change. On Day 11 metaphases were present in the interstitial tissue which regenerated on Day 28. Between Days 5 and 7 some pycnotic nuclei of spermatocytes appeared, mitotic activity of spermatogonia was normal, but germ cell stages were disorganized and empty spaces appeared at the boundary of the tubule. These data show that a single EDS injection results in destruction and repopulation of the interstitial cells in a reptile. Moreover, the effects of EDS in the lizard suggest that P. s. sicula Raf testis responds to the toxin in a similar fashion to the rat testis.

Androgens↗

PubMed1995

Changes in proto-oncogene activity in the testis of the frog, Rana esculenta, during the annual reproductive cycle.

Proto-oncogenes are said to influence the regulation of cellular growth and differentiation. Myc, Fos, Jun, and Mos protein localization has been studied by immunocytochemistry in the testis of the frog, Rana esculenta, during the annual reproductive cycle. Oncoproteins have been localized in the primary and secondary (I and II) spermatogonia (SPG). Myc and Mos also appear in I and II spermatocytes (SPC) while Jun appears in II SPC. Myc, Fos, and Jun in SPG translocate in the nucleus during the periods of active spermatogenesis. Myc, Fos, and Jun are also localized in Sertoli cells. Fos is present in interstitial cells during the period characterized by the androgen peak which precedes the sharp increase of estradiol. It is suggested that proto-oncogene activity exerts a regulatory role in steroidogenesis and spermatogenesis.

Androgens↗

PubMed1995

Organogenesis of the orbital glands in the lizard Podarcis s. sicula: a histological, histochemical and ultrastructural study.

The orbital glands of the lizard Podarcis s. sicula are represented by the anterior and posterior lacrimal glands and the Harderian gland. The anlage of the Harderian gland appears on about the 22nd day of development in the form of a short tubule projecting from the conjunctival epithelium. This event is coincident with the appearance of the nictitating membrane. At this stage the mesenchymal cells surrounding the glandular blastema proliferate at a high rate and form a definite sac, later occupied by both the Harderian gland and the anterior lacrimal glands. At the 26th day of development, the glandular blastema forms acini at its distal end. The prospective glandular cells are not yet differentiated histologically. At the 36th day of development, differentiated serous glandular cells become visible. At the 41st day of development, the acini fill up the preformed mesenchymal sac. Only at this stage does the most medial part of the gland differentiate into mucous-secreting anterior lacrimal gland. At the same time, a small primordium of the posterior lacrimal gland can be seen in the posterior commissure of the eye. The appearance of junctional complexes between epithelial cells and mesenchymal cells in the early developmental stages supports the role of the mesenchyme in the differentiation of the glandular cells. Since the glandular anlage differentiates laterally into Harderian gland and medially into anterior lacrimal gland, spatial and temporal differences seem to exist in the inductive process. Furthermore, a concentration gradient of the inductive substance(s) may be envisaged, since an intermediate zone is present between the Harderian gland and the anterior lacrimal gland, consisting of mixed glandular cells containing both mucous and serous secretory granules.

Animals↗

PubMed1995

Effect of cholinergic secretagogue substances on the morphology of the harderian gland in the frog, Rana esculenta.

The present study using secretagogue substances was undertaken to investigate Harderian gland secretion in the frog, Rana esculenta. Carbamylcholine chloride and bethanechol injections caused enhancement of the secretory activity and hyperemia, while nicotine did not. Morphological examinations showed reduced cellular height and dilated alveolar lumina, containing secretory granule discharge, nuclei and cytoplasmic fragments, indicating an apocrine and holocrine secretion type. The administration of atropine prevented the enhancement of the secretion. Our data suggest that cholinergic stimulation provokes enhancement of the secretory activity on the frog HG, and this mechanism appears to be mediated by the activation of the muscarinic receptors.

Animals↗

PubMed1995

Mast cell-Leydig cell relationships in the testis of the lizard Podarcis s. sicula Raf: thermal manipulation, ethane 1,2-dimethane sulphonate (EDS) and sex hormone treatment.

Mast cells of connective tissue type are scattered in the interstitial compartment of the lizard Podarcis s. sicula. Their number varies during the year, showing peaks in spring and in winter, respectively. Thermal manipulation affects mast cell number (MCN): high temperature decreased MCN in both January and May, while low temperature increased MCN only in January. Ethane dimethane sulphonate, a toxin which specifically destroys Leydig cells, induced an increase in MCN on days 3 and 7 of treatment. Oestradiol treatment provoked a strong increase in MCN that was blocked by tamoxifen. Blocking androgen receptors with cyproterone acetate resulted in an increase in MCN, while testosterone injection provoked a strong decrease. These results suggest a relationship between the presence of mature Leydig cells and mast cell proliferation and/or differentiation.

Androgen Antagonists↗

PubMed1995

The effects of testosterone and estradiol on mast cell number in the harderian gland of the frog, Rana esculenta.

The Harderian gland (HG) of the frog Rana esculenta contains mast cells in the interstitial tissue. The mast cell number (MCN) is influenced by sex hormones. Gonadectomy in both sexes provoked a decrease in MCN in January, while no effect was observed in September. Sex hormone-replacement therapy gave different results; estradiol treatment in castrated males and females always increased MCN, while testosterone did not. Acute estradiol treatment provoked an increase in MCN on days 2 and 4 of treatment and the morphology of the glandular compartment appeared normal. On days 8, 10 and 12 of treatment the MCN drastically decreased. The majority of glandular acini appeared strongly disorganized and the interstitial tissue became hypertrophic in concomitance with an increased vascularization. Our results suggest that estradiol acts by stimulating mast cells and acute estradiol treatment provokes proliferation of interstitial connective tissue together with glandular cells damage.

Animals↗

PubMed1994

Regeneration of the testicular interstitial compartment after ethane dimethane sulfonate treatment in the hypophysectomized frog Rana esculenta: independence of pituitary control.

The effects of ethane dimethane sulfonate (EDS) on the testes of hypophysectomized frogs (Rana esculenta) were investigated by light and electron microscopy. Initial signs of interstitial cell damage were observed in EDS- and EDS plus pituitary homogenate (PH)-treated animals 5 days after a single injection of EDS (100 mg/Kg body weight). The germinal compartment in these two groups appeared disorganized adjacent to the damaged interstitial tissue only in the EDS-treated animals, and by Day 8, spermatogenesis seemed to be affected in the EDS + PH-treated frogs in which Leydig cells had disappeared in some areas. On Day 28, regeneration of the interstitial tissue was complete and spermatogenesis was restored to normal. These data suggest that, in hypophysectomized frogs, the regeneration of the interstitial compartment is independent of pituitary activity and that the lack of interstitial cells activates the production of local factors responsible for the differentiation and proliferation of new Leydig cells. It is concluded that in addition to gonadotropins, the intratesticular environment is fundamental in the maintenance and regulation of testicular structure and function.

Androgens↗

PubMed1994

The effect of sex hormones on lipid content and mast cell number in the harderian gland of the female toad, Bufo viridis.

The Harderian gland of the toad Bufo viridis is a dimorphic gland owing to the presence of lipid droplets in the female glandular cells present only during summer months. Ovariectomy causes the disappearance of sudanophilia and estrogen-treatment completely prevents this change, while testosterone-injection has little effect. Estradiol-treatment also provokes a proliferation of the interstitial connective tissue concomitantly with the mast cell number increase. Our results suggest that estradiol acts, stimulating both mast cell and connective tissue proliferation, and plays a role in determining the expression of the female type of the toad Harderian gland.

Animals↗

PubMed1994

Quantitative increases in DNA binding affinity and positional effects determine 9-cis retinoic acid induced activation of the retinoid X receptor beta homodimer.

Retinoid X receptors (RXRs) exert transcriptional activities through heterodimerization with members of the nuclear hormone receptor superfamily. RXRs also act as homodimers and stimulate transcription from an RXR responsive element (RXRE) when bound to 9-cis-retinoic acid (9cRA). Here direct effects of 9cRA have been examined on biochemical and mechanistic parameters of RXR beta. It is shown that 9cRA significantly increases RXR beta homodimer binding affinity to an RXRE (Kd without ligand = 18 nM, Kd with ligand = 6 nM), while decreasing significantly the affinity of RXR beta/thyroid hormone receptor (T3R alpha) heterodimer binding to the same element. Effects on other response elements are also examined. The RXR beta homodimer was found to contact both halves of the RXRE direct repeat, irrespective of the effect of added ligand, while the RXR beta/T3R alpha heterodimer contacted the element only through a specific half-site. Binding of the homodimer to the element functionally activates RXR beta, since RXR beta enhanced transcription in vitro from a specific template in a ligand-dependent fashion. In agreement, transfection of RXR beta alone (but not RXR beta/T3R alpha) led to ligand-dependent activation of a reporter containing the RXRE. Taken together, 9cRA facilitates functional activation of the RXR beta homodimer in an element-dependent manner.

Animals↗

PubMed1994

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Animals↗

PubMed1994

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

Animals↗

PubMed1994

T cell activation and increases in protein kinase C activity enhance retinoic acid-induced gene transcription.

Retinoic acid (RA) has profound effects on cell growth and differentiation. Its receptors are members of the steroid/thyroid hormone receptor superfamily, which regulates nuclear transcription and gene expression by binding specific response elements. Protein kinase C (PKC) is activated during signal transduction initiated by a variety of membrane receptors. Using a RA-responsive element and reporter gene construct transfected into a T cell, we found: 1) T cell activation and PKC activators enhance transactivation by RA, 2) down-regulation of PKC protein has little effect on RA transactivation but abolishes superinduction by phorbol ester, which is restored by cotransfection of a PKC alpha-expression vector, and 3) cotransfection of dominant-negative c-jun does not prevent superinduction by phorbol ester. Together, these data demonstrate that PKC can modulate RA signal transduction, apparently without involvement of AP-1, and provide a new example of cross-talk between signal transduction pathways.

Animals↗

PubMed1994

Regional and seasonal variations of RNA synthesis in the brain of the green frog, Rana esculenta.

Changes of RNA synthesis were demonstrated in neurons and ependymal cells of the green frog Rana esculenta during the annual cycle using the Mallory's trichrome stain as histochemical marker and autoradiography. Since the higher affinity of the nuclei for aniline blue is consistent with the increase of RNA content, the increase of RNA synthesis was expressed as percentage of the blue stained nuclei (% BSN). Neuronal transcription starts slowly in March or April, reaches a maximum in July and declines from September to November or December, depending on the brain region. In the ependymal cells, RNA synthesis starts in March and lasts until October. Neuronal transcriptional activity is found mostly in the glomerular layer of the olfactory bulb, in the striatum, nucleus accumbens septi, lateral and medial septal nuclei of the telencephalon, in the habenulae and various nuclei of the diencephalon, in the tectum opticum (particularly in the stratum griseum centrale), in the molecular layer of the cerebellum and in various nuclei of the rhombencephalon. The transcriptional activity of the ependymal cells is quite uniform in the lateral ventricles and the fourth ventricle, while it shows regional symmetric distribution in the third ventricle. Seasonal differences in transcriptional activity appear to be independent of seasonal thermic and photoperiodic fluctuations. In fact, temperature and photoperiod manipulations do not modify significantly the number of active nuclei. It is likely that the increase of RNA synthesis in nerve and ependymal cells corresponds to the resumption of neurotransmitter biosynthesis after hibernation. The simple Mallory's trichrome stain provides a reliable method for revealing increased transcriptional activity in histological sections.

Animals↗

PubMed1993

Dopamine regulation of testicular activity in intact and hypophysectomized frogs, Rana esculenta.

In intact frogs, both GnRHA and L-dopa were able to increase testicular and plasma androgen levels and to induce spermiation. The dopamine antagonist pimozide inhibited both the effects of L-dopa but not those of GnRHa. Hypophysectomy reduced androgen levels, but spermiation was still induced by both GnRHa and L-dopa, suggesting that these agents can directly influence the testis through a route not involving the pars distalis. Again, pimozide antagonised spermiation induced by L-dopa but not that induced by GnRHa.

Androgens↗