Betamethasone, progesterone and RU-486 (mifepristone) exert similar effects on connexin expression in trophoblast-derived HTR-8/SVneo cells.

Connexins (Cx) are membrane proteins able to influence cell trophoblast responses, such as proliferation, differentiation, migration and invasiveness. Likewise, glucocorticoids are also known to modulate many factors involved in implantation, including trophoblast gap-junction intercellular communication, although their influence on pregnancy is controversial. In order to investigate the effects of betamethasone, a synthetic glucocorticoid, on Cx and glucocorticoid receptor (GR) expression and localisation, as well as on cell proliferation, the extravillous trophoblast-derived HTR-8/SVneo cell line was used as a model. The results, confirmed by means of immunofluorescence, demonstrate that betamethasone selectively modifies GR and Cx expression, enhancing the GRα isoform without affecting GRß, and inhibiting Cx40 expression whilst increasing that of Cx43 and Cx45. Furthermore, betamethasone was shown to exert an inhibitory action on cell proliferation. In this model the abortion drug RU-486 (mifepristone), reported to be a GR antagonist, did not counteract this effect of betamethasone. On the contrary, it induced responses similar to those of the hormone. Knowing that RU-486 is also a potent progesterone-receptor antagonist, the effect of progesterone alone and in combination with the drug on Cx expression and cell proliferation was then tested. Progesterone showed the same effect as betamethasone on Cx expression, but it did not affect proliferation. Based on these results, neither the abortion effects of RU-486 nor the protective action of betamethasone and progesterone are exerted by modulation of Cx. RU-486 did not antagonise the progesterone effect, suggesting that its abortive action does not involve alteration of trophoblast Cx expression.

cAMP efflux from human trophoblast cell lines: a role for multidrug resistance protein (MRP)1 transporter.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is a second messenger, which exerts an important role in the control of human first-trimester trophoblast functions. In the present study we demonstrate the existence of a mechanism that is able to extrude cAMP from trophoblast-derived cell lines, and show evidence indicating the involvement of multidrug resistance protein (MRP) 1, a transporter belonging to the ATP-binding cassette family, in cAMP egress. MRP1 is expressed in trophoblast cell lines and cAMP efflux is highly reduced by the MRP1 inhibitor, MK-571. In addition, interleukin-1beta and estrone are able to enhance MRP1 gene expression and influence extracellular cAMP concentration. The occurrence of a MRP1-dependent cAMP efflux is also shown in human first-trimester placenta explants. Extracellular cAMP could represent a source for adenosine formation, which in turn could regulate cAMP-dependent responses in placental tissue. Evidence is provided that adenosine receptor subtypes are present and functional in human trophoblast-derived cells. A role for cAMP egress mechanism in the fine modulation of the nucleotide homeostasis is therefore suggested.

Lack of augmentation of tumor spectrum or severity in dual heterozygous Men1 and Rb1 knockout mice.

To identify possible genetic interactions between the mechanisms of tumor suppression of menin and pRb, we intercrossed mice with targeted deletions of Men1 and Rb1, and compared tumor development in cohorts of animals carrying single or dual mutations of these tumor-suppressor genes. In mice lacking one copy of Men1, pancreatic islet and anterior pituitary adenomas are common. In animals lacking one copy of Rb1, intermediate pituitary and thyroid tumors occur at high frequency, with less frequent development of pancreatic islet hyperplasia and parathyroid lesions. In mice heterozygous for both Men1 and Rb1, pancreatic hyperplasia and tumors of the intermediate pituitary and thyroid occurred at high frequency. Serum measurements of calcium and glucose did not vary significantly between genotypic groups. Loss of heterozygosity at the Rb1 locus was common in pituitary and thyroid tumors, whereas loss of menin was observed in pancreatic and parathyroid lesions. The tumor spectrum in the double heterozygotes was a combination of pathologies seen in each of the individual heterozygotes, without decrease in age of onset, indicating independent, non-additive effects of the two mutations. Together with the lack of increased tumor spectrum, this suggests that menin and pRb function in a common pathway of tumor suppression.

Effects of cadmium on MAPK signalling pathways and HSP70 expression in a human trophoblast cell line.

The aim of this work was to provide a greater insight into the possible effects of Cd on signal transduction and stress-related pathways in reproductive tissues. Cd is a known placental toxin in both animals and humans. Our experiments were designed to study the influence of Cd on MAPK (ERK1/2, JNK1/2 and p38MAPK) activation in the extravillous trophoblast cell line, HTR-8/SVneo, used as an experimental model. We also studied the HSP70 response in cells exposed to Cd, since these proteins may have an important role in conferring protection and tolerance against teratogenic concentrations of the metal. The effects of Cd were compared with those of a well-known toxic agent, H2O2. The metal triggered MAPK activation in a dose- and time-dependent manner. At 30 microM Cd, stimulations of about 300%, 550% and 250% were observed for ERK1/2, JNK1/2, and p38MAPK, respectively. Phosphorylation of ERK1/2 and JNK1/2 was significantly induced after a 1-h exposure to 30 microM Cd, while that of p38MAPK occurred only after 8h. Similarly, H2O2 caused dose- and time-dependent activation of MAPK pathways. Cd potently stimulated HSP70 expression and that of related genes HSP70 A, B and C. H2O2 did not increase HSP70 and HSP70 A and B expression, while temporarily increasing HSP70C transcript levels. In conclusion, Cd triggers different stress responses in trophoblast cells involving HSP70 and SAPK, and also enhances ERK1/2 phosphorylation. Since MAPK dependent pathways play a crucial role during pregnancy, non-physiological activation by Cd exposure may disrupt normal functions in trophoblast cells.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate.

We have previously demonstrated that dopamine conjugation to glucose allows it to induce therapeutic effects against Parkinson's disease after intravenous administration. In this paper we demonstrate that, unlike dopamine, the prodrug glu-dopamine is a transportable substrate of glucose transporters. Towards this, the effect of glucose-conjugation on the affinity and uptake of dopamine have been assessed in vitro, using human retinal pigment epithelium (HRPE) cells. Glucose transporter-mediated uptake was measured using [(3)H]3-O-methylglucose ([(3)H]3-O-MG) as the tracer. The uptake was found to be rapid and hyperbolically related to its concentrations (K(t)=7.8+/-1.2mM and V(max)=54+/-2 nmol/min mg protein). Inhibition experiments showed that dopamine was able to interact with glucose carriers only when conjugated to glucose (IC(50)=2.6+/-0.6mM). HPLC analysis of HRPE cell extracts showed that both dopamine and the prodrug permeate the cell, but only the uptake of the prodrug is inhibitable by glucose. This confirms that glucose transporters mediate the transport of the prodrug glu-dopamine, but not of dopamine. HRPE cells is therefore proposed as a promising model for in vitro studies involving the glucose transporter-mediated transport of drugs and their conjugates.

Nuclear retinoic acid receptor beta as a tool in chemoprevention trials.

Retinoic acid (RA) and its natural and synthetic derivatives (retinoids) are important dietary factors which regulate cellular differentiation and growth, so that they are thought to be particularly effective at preventing the development of several tumours. They play this role as ligands of the RAR and RXR nuclear retinoic acid receptors, including the RA receptor isoforms alpha, beta, and gamma. These ligand-activated nuclear receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. Among these target genes, the RARbeta gene is of great interest, being able to encode a potential tumour suppressor. It should be emphasized that most breast carcinomas and breast cancer cell lines show loss or down-regulation of RARbeta receptor expression, whereas RARalpha and gamma, as well as retinoid X receptors, appear to be variably expressed in both normal and tumour cells. It is also interesting to note that basal and RA-induced RARbeta mRNA levels tend to increase with senescence of normal cells. This information provides further support for the hypothesis that genetic events involved in cellular senescence may also play a significant role in tumour suppression in humans. The aim of this review is to clarify whether expression of RARbeta could be modulated by chemopreventive intervention and may therefore serve as an intermediate biomarker in chemoprevention trials for some cancers.

Prostaglandin E2 inhibits proliferation and migration of HTR-8/SVneo cells, a human trophoblast-derived cell line.

Normal placentation requires a highly coordinated control of proliferation, migration and invasiveness of extravillous trophoblast cells. Since prostaglandin E2 is a major prostanoid synthesized by intrauterine tissues and highly involved in pregnancy homeostasis, we examined the possibility that it modulates extravillous trophoblast cell functions. Here, we report the presence of mRNAs for prostaglandin E2 EP2 and EP4 receptor isoforms and of proteins in both first-trimester human chorionic villi and in the human trophoblast-derived HTR-8/SVneo cells. Moreover we found that: (i) this cell line releases prostaglandin E2 and the output is enhanced by interleukin-1beta; (ii) the prostanoid consistently inhibits serum- or epidermal growth factor-induced cell proliferation and also migration. An involvement of cAMP in the prostaglandin E2 antiproliferative action is suggested by the observation that the prostanoid greatly enhances cAMP level in HTR-8/SVneo cells and that forskolin inhibits cell proliferation; moreover the administration of prostaglandin E2 plus forskolin, a condition which evokes a synergistic enhancement of cAMP, induces a major impairment of cell growth. Provided that our data are applicable to the trophoblast tissue in vivo, we suggest that prostaglandin E2 exerts an important control on extravillous trophoblast cell functions, preventing an excessive proliferation and migration.

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes.

Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDS-PAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis.

Two for-Met-Leu-Phe-OMe analogues trigger selective neutrophil responses. A differential effect on cytosolic free Ca2+.

For-Thp-Leu-Ain-OMe and for-Met-delta(z)Leu-Phe-OMe are two conformationally restricted fMLP-OMe analogues able to discriminate between different biological responses of human neutrophils. In this paper, we demonstrate that the former peptide, which evokes only chemotaxis, does not alter human neutrophil Ca2+ levels. In contrast, for-Met-delta(z)Leu-Phe-OMe, which induces superoxide anion release and degranulation but not chemotaxis, significantly increases the cation concentration. The chelation of Ca2+ in both extracellular and intracellular media abolishes O2- production triggered by for-Met-delta(z)Leu-Phe-OMe, while the same procedure does not affect neutrophil chemotaxis towards for-Thp-Leu-Ain-OMe. We therefore suggest that chemotaxis, unlike superoxide anion release, is independent of Ca2+ enhancement in human neutrophils.

Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues.

The N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-OMe (1) analogues for-Thp-Leu-Ain-OMe (2), for-Thp-Leu-Phe-OMe (3), for-Met-Leu-Ain-OMe (4), for-Met-Delta(z)Leu-Phe-OMe (5), for-Met-Lys-Phe-For-Met-Lys-Phe (6), for-Met-Leu-Pheol-COMe (7), and for-Nle-Leu-Phe-OMe (8) have been studied. Some of these have been found selective towards the activation of different biological responses of human neutrophils. In particular, peptides 2 and 3, which evoke only chemotaxis, are ineffective in enhancing inositol phosphate, as well as cyclic AMP (cAMP) levels. On the contrary, analogues 5 and 7, which induce superoxide anion production and degranulation, but not chemotaxis, significantly increase the levels of the two intracellular messengers, as is the case of the full agonists 1 and 6. The Ca(2+) ionophore A23187 also activates phospholipase C (PLC) and increases the nucleotide levels; when tested in combination with peptide 1 or 5, a supra-additive enhancement of cAMP concentration is obtained. The PLC blocker, U-73122, inhibits the formylpeptide-induced inositol phosphate formation, as well as cAMP increase. Moreover, this drug drastically reduces superoxide anion release triggered by 1 or 5, whereas it inhibits to a much lesser extent neutrophil chemotaxis induced by 1 or 2. Our results suggest that: (i) PLC stimulation is involved in cAMP enhancement by formylpeptides; (ii) the activation of PLC by formylpeptides, in conditions of increased Ca(2+) influx, induces a supra-additive enhancement of the nucleotide; (iii) the inability of pure chemoattractants to significantly alter the PLC activity or cAMP level, differently from full agonists or peptides specific in inducing superoxide anion release, appears as a general property. Thus, the activation of neutrophil PLC seems essential for superoxide anion release, but less involved in the chemotactic response.

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides.

The increase in human neutrophil cyclic adenosine monophosphate (cAMP) levels evoked by formylated peptides is significantly reduced in the presence of MDL 12330A, SQ 22536, GDPssS and clonidine, which inhibit the adenylyl cyclase system by acting at different sites in this enzyme complex. A similar effect is exerted by adenosine deaminase and dipyridamole, which alter the extracellular adenosine concentration. Neutrophil preincubation with adenylyl cyclase inhibitors or dipyridamole reduces chemotaxis and superoxide anion production triggered by peptides; adenosine deaminase, on the contrary, has no effect on neutrophil responses. Our results seem to indicate that: (1) the peptide-induced increase in neutrophil cAMP is due mainly to an action on the adenylyl cyclase system; (2) an enhancement of this cyclic nucleotide, even slight and necessarily transient, is required for chemotaxis and O2 production induced in neutrophils by formylated peptides; and (3) cAMP does not represent the crucial second messenger for adenosine in the modulation of neutrophil responses.

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues.

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.

Vitamin C and 6-amino-vitamin C conjugates of diclofenac: synthesis and evaluation.

Diclofenac (Diclo), its ascorbic acid (AA) or 6-amino-AA (AA-NH2) pro-drugs (AA-Diclo or AA-NH-Diclo) were prepared and evaluated on human retinal pigment epithelium (HRPE) cells to investigate their ability to interact with the vitamin C transporter SVCT2 and their cellular uptake. Furthermore, stabilities in physiological fluids of these compounds were investigated. For kinetic experiments, AA-Diclo was incubated in Tris-HCl buffer, human plasma or whole blood. The extracted samples were analysed by HPLC. AA-Diclo was hydrolysed following first order kinetics in buffer, plasma (t1/2 about 10 h) and whole blood (t1/2 about 3.5 h). Transport and inhibition assays were performed by adding [14C]AA and the above-mentioned unlabelled compounds to plated HRPE cells. Intracellular accumulation was measured incubating HRPE cells with increasing concentrations of unlabelled compounds, following by HPLC analysis. Diclo resulted as a non-competitive inhibitor of AA-transport, showing a Na+-dependent and ascorbate-independent uptake. AA-Diclo behaved as a competitive inhibitor, but it was not transported into cells, whereas its analogue AA-NH-Diclo showed a decreased inhibitory activity. Stability studies suggest AA-Diclo as a potential candidate to enhance the Diclo short half life in vivo. The discovery of a Na+-dependent transporter for Diclo on HRPE cells opens new perspectives for targeting diclofenac into the brain.

Increased oxidative modification of albumin when illuminated in vitro in the presence of a common sunscreen ingredient: protection by nitroxide radicals.

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

Biological activity of somatostatin receptors in GC rat tumour somatotrophs: evidence with sst1-sst5 receptor-selective nonpeptidyl agonists.

The physiological actions of somatostatin-14 (SRIF: somatotrophin release inhibitory factor) receptor subtypes (sst(1)-sst(5)), which are endogenously expressed in growth cells (GC cells), have not yet been elucidated, although there is evidence that sst(2) receptors are negatively coupled to cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and adenosine 3,5'-cyclic monophosphate (cAMP) accumulation. In addition, both sst(1) and sst(2) receptors are negatively coupled to growth hormone (GH) secretion in GC cells. Here we report on studies concerning the expression, the pharmacology and the functional role of native SRIF receptors in GC cells with the use of five nonpeptidyl agonists, highly selective for each of the SRIF receptors. Radioligand binding studies show that sst(2) and sst(5) receptors are present at different relative densities, while the presence of sst(3) and sst(4) receptors appears to be negligible. The absence of sst(1) receptor binding was unexpected in view of sst(1) receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low-density population of sst(1) receptors. Functionally, only sst(2) receptors are coupled to the inhibition of [Ca(2+)](i) and cAMP accumulation and the selective activation of sst(5) receptors facilitates the stimulation of adenylyl cyclase activity through G(i/o) proteins. This effect was not observed when sst(2) and sst(5) receptors were simultaneously activated, suggesting that there is a functional interaction between sst(2) and sst(5) receptors. In addition, sst(1), sst(2) and sst(5) receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca(2+)](i) and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provides compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity.

Somatostatin-induced control of cytosolic free calcium in pituitary tumour cells.

1. In rat pituitary tumour cells (GC cells), spontaneous oscillations of the intracellular concentration of Ca2+ ([Ca2+]i) induce growth hormone (GH) secretion that is inhibited by octreotide, a somatostatin (SRIF) agonist which binds to SRIF subtype (sst) receptor 2. The effects of its functional activation on the control of [Ca2+]i were investigated using fluorimetric measurements of [Ca2+]i. 2. SRIF decreases the basal [Ca2+]i and the [Ca2+]i rise in response to forskolin (FSK) through the inhibition of L-type voltage-dependent Ca2+ channels. 3. Pretreatment with octreotide or with L-Tyr8++ Cyanamid 154806, a sst2 receptor antagonist, abolishes the SRIF-induced inhibition of [Ca2+]i. Octreotide is known to operate through agonist-induced desensitization, while the antagonist operates through receptor blockade. 4. sst1 and sst2 receptor-immunoreactivities (-IRs) are localized to cell membranes. sst2, but not sst1 receptor-IR, internalizes after cell exposure to octreotide. 5. SRIF-induced inhibition of basal [Ca2+]i or FSK-induced Ca2+ entry is blocked by pertussis toxin (PTX). 6. FSK-induced cyclic AMP accumulation is only partially decreased by SRIF or octreotide, indicating that sst2 receptors are coupled to intracellular pathways other than adenylyl cyclase (AC) inhibition. 7. In the presence of H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA), SRIF-induced inhibition of basal [Ca2+]i is still present, although reduced in amplitude. 8. SRIF inhibits [Ca2+]i by activating sst2 receptors. Inhibition of AC activity is only partly responsible for this effect, and other transduction pathways may be involved.

Effect of nitric oxide on arachidonic acid release from human amnion-like WISH cells.

In order to clarify the possible interactions between nitric oxide (NO) and arachidonic acid (AA) pathways, human amnion-like WISH cells were perifused to measure the effects of the following substances on [(3)H]arachidonic acid release: (1) sodium nitroprusside (SNP), a nitric oxide donor; (2) 1,1,1-trifluoromethyl-6,9,12,15-heicosatetraen-2-one, a cytosolic phospholipase A(2) (cPLA(2)) inhibitor; (3)L -arginine, the substrate of nitric oxide synthase (NOS); (4) 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole and 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, activator and inhibitor of soluble guanylyl cyclase, respectively; (5) a membrane-permeable non-hydrolyzable analogue of guanosine-3',5'-cyclic monophosphate (cGMP). Furthermore, the effect of SNP on prostaglandin E(2) (PGE(2)) release was tested. Exogenous and endogenous NO, as well as the guanylyl cyclase activator and cGMP analogue, significantly increased [(3)H]arachidonic acid release. Both soluble guanylyl cyclase and PLA(2) inhibitors counteracted SNP response. Exogenous NO increased PGE(2) release, although to a much lesser degree compared with arachidonic acid release. Our results indicate that NO stimulates AA release in WISH cells by activating PLA(2) through a cyclic GMP-dependent mechanism.

Premature ovarian failure (POF) and fragile X premutation females: from POF to to fragile X carrier identification, from fragile X carrier diagnosis to POF association data.

Early menopause in the fragile X carriers has been well documented in several reports. All surveys demonstrated that 13-25% of fragile X carriers experienced premature ovarian failure (POF), defined as menopause before the age of 40 years. In 1995 we started screening two groups of subjects as a part of a Fragile X Research Program: 1) women previously diagnosed as fragile X carriers from the register of our center and 2) women with POF and without a family history of fragile X or other forms of mental retardation. In this study we report the preliminary data collected from 75 fragile X families; in 30 of them, POF was present in one or several subjects, all of whom had a fragile X premutation. None of the women with a full mutation experienced POF in our series of patients. We also identified 89 families without a family history of fragile X or mental retardation, and there were 108 subjects who experienced POF, of which 6.5% had a fragile X premutation. This is 70-fold higher than the background prevalence of fragile X premutation in the Italian population and suggests an association with POF. These data confirm the results of other surveys.

WISH cells as a model for the "in vitro" study of amnion pathophysiology.

In the course of pregnancy amnion cells produce a number of factors which include cytokines and prostaglandins (PGs) produced in response to autocrine, paracrine and endocrine signals. Recent studies performed by several researchers contributed to elucidate the mechanism through which amnion tissue is involved in the triggering of physiological labor. However, there are other possible functions to be ascribed to amniotic cells, depending on the high number of factors that they produce as well as on the receptors that enable them to act in turn as target. For instance, it has been demonstrated that amnion cells are able to produce lecithin upon the regulation of several factors, such as glucocorticoids and epidermal growth factor, a finding that suggests a protective role of the tissue on fetal pulmonary function. As regards to triggering the uterine contractions, it is accepted that prostaglandin release by amnion cells represents a key event. It is under the control of hormones, growth factors, cytokines and probably PGs themselves. A striking analogy has been found between the mechanism of inflammation and the onset of myometrial activity in labor. In this context, it has been shown that for-Met-Leu-Phe (fMLP), the prototype of a series of formylated peptides traditionally considered chemotactic agents, is also involved in the regulation of amniotic PG release. The similitude between labor and inflammatory response is enforced by the antiprostaglandin action of some classes of antibiotics observed in amnion tissue, that enable them as effective tools against preterm labor, both in the absence and in the presence of infection. As for the mechanisms responsible for the regulation of PG synthesis, some agents act by influencing protein synthesis, while others exert their effects through the production of intracellular second messengers, mainly represented by phosphatidyl-inositol-4-5 bisphosphate and cyclic AMP. The mechanism whereby second messengers induce PG release is not clear, and a crosstalk between the two transduction pathways could be hypothesized. This interaction has extensively been analysed in "WISH" cells, a human amnion-derived cell line, which represent a model for the in vitro study of amnion functions. In the present review, we intend to report the results of the studies regarding the mechanisms through which the control of the above mentioned functions is executed.