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Biomedical subjects

M Di Domenico

Publications and source records attributed to M Di Domenico.

At least 19 recordsLinked to original sources
PubMed2006

p85 regulatory subunit of PI3K mediates cAMP-PKA and estrogens biological effects on growth and survival.

Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.

Animals↗
PubMed2002

PANSS factors and scores in schizophrenic and bipolar disorders during an index acute episode: a further analysis of the cognitive component.

To investigate the factor structure of psychotic symptoms, we compared the clinical characteristics of manic patients with those of schizophrenic patients evaluated with positive and negative syndrome scale (PANSS). The clinical symptoms of 148 bipolar patients and 86 schizophrenic patients hospitalized for an index psychotic episode were assessed. Schizophrenic patients showed more positive and cognitive symptoms than bipolars. The factor analysis of the two PANSS scores showed a three-factor solution with 'positive', 'negative' and 'mixed' depressive-activated factors for bipolars and 'positive', 'negative' and 'depressive' factors for schizophrenics. In both groups, the 'cognitive cluster' loaded on the first 'positive' factor while the 'lack of insight' (LOI) has a different meaning in the two groups, more related to the positive symptoms in the bipolar patients and more related to the negative symptoms in the schizophrenic patients. This finding suggests that LOI could be a non-unitary phenomenon in psychoses and it should be further explored to better elucidate differences in symptom structures between schizophrenic and bipolar disorders.

Acute Disease↗
PubMed2002

Sex steroid hormones act as growth factors.

We observed that sex steroid hormones, like growth factors, stimulate the Src/Ras/erk pathway of cell lines derived from human mammary or prostate cancers. In addition, hormone-dependent pathway activation can be induced in Cos cells, upon transfection of classic steroid receptors. Cross-talks between sex steroid receptors regulate their association with Src and consequent pathway activation. Oestradiol treatment of MCF-7 cells triggers simultaneous association of ER with Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase (PI3-kinase) and activation of Src- and PI3-K-dependent pathways. Activation of the latter pathway triggers cyclin D1 transcription, that is unaffected by Mek-1 activation. This suggests that simultaneous activation of different signalling effectors is required to target different cell cycle components. Thus, a novel reciprocal cross-talk between the two pathways appears to be mediated by the ER. In all tested cells, activation of the signalling pathways has a proliferative role. Transcriptionally inactive ER expressed in NIH 3T3 cells responds to hormone causing Src/Ras/Erk pathway activation and DNA synthesis. This suggests that in these cells genomic activity is required for later events of cell growth.

3T3 Cells↗
PubMed2002

Src is an initial target of sex steroid hormone action.

Recent observations that steroids use pathways universally known to be regulated by growth factors and interleukins highlight the following points: (1) Steroid stimulation of the canonical pathway Src/Ras/Erk signaling from membrane to nuclei or its single members has been observed in different cell types including human cancer-derived cells, neurons, osteoblasts, osteocytes, and endothelial cells. This stimulation has been reconstituted and analyzed in transiently transfected cells. (2) Cellular context and intracellular localization of receptors are crucial in determining the biological effects evoked by this hormonal stimulation: proliferation, protection from apoptosis, and vasorelaxation. (3) Classical steroid receptors localized in the extranuclear compartment directly and, in some cases, simultaneously interact with Src. They are capable of unexpected cross talks responsible for the observed effects. (4) Other signaling pathways including P13K/AKT are also stimulated by steroids. The aim of future work will be to arrive at an integrated general view of the different signaling pathways activated by steroids and to analyze the concert between these pathways and the hormonal transcriptional action. This general view should be simultaneously verified in different cell contexts, under different physiologic and pathologic conditions. We expect that the new technologies, above all gene and protein microarray, will make this goal feasible.

Apoptosis↗
PubMed2002

Fertility rate evaluation by laparoscopic approach in the experimental animal.

PURPOSE OF INVESTIGATION: The aim of the present study was to evaluate the effect of laparoscopic insemination (LAP) and natural mating (NM) on fertility rate in experimental animal (Ovis Aries Comisana) during the month of June. METHODS: For the experiment, 97 ewes were used. Laparoscopic insemination was performed with the frozen semen of three different Romanov rams: Laparoscopic insemination I (n = 24); Laparoscopic insemination 2 (n = 26); and laparoscopic insemination 3 (n = 28), and natural mating was performed with two different Ovis Aries Comisana rams with proven fertility: Natural mating I (n = 10); Natural mating 2 (n = 9). Estrus was synchronized with fluorogestone acetate impregnated intravaginal sponges (40 mg, 14 days). Pregnant mare serum gonadotrophin (Folligon, Intervet International) at a dose of 400 UI was given intramuscularly at sponge removal. Artificial insemination was carried out 60 hours after the removal of the sponges in the laparoscopic insemination groups. RESULTS: The mean pregnancy rate at ecographic diagnosis performed at about 36 days from sponge removal for the laparoscopic insemination and natural mating groups were respectively, 62.8% and 78.9% with no significant difference. CONCLUSION: The mean fertility rates for the LAP and NM groups were 56.0 and 73.4, respectively, with no significant difference.

Animals↗
PubMed2002

Ovine fetal growth curves in twin pregnancy: ultrasonographic assessment.

PURPOSE OF INVESTIGATION: The fetal-lamb model is a fundamental tool for developing clinical applications for the treatment of human fetal pathology (e.g., open fetal surgery). Accurate estimation of gestational age is important to avoid size-related problems during surgery. METHODS: To evaluate the effect of twinning on ovine fetal growth, we followed seven twin pregnancies in Comiso ewes from mating through parturition. Fetal growth indexes (muzzle-occipital and mean abdominal diameters, abdominal circumference, femoral and humeral length) were measured weekly using perimammary ultrasonography and analyzed with a linear quadratic regression model based on natural logarithms of each parameter and fetal age. RESULTS: The model explained > 90% of the variability observed, with determination coefficients of 95% (femoral length, abdominal circumference), 94% (abdominal diameter, humeral length), and 89% (muzzle-occipital diameter). CONCLUSION: Mean birth weight was lower than that reported for singleton fetal lambs, as it is in bigeminal pregnancies in humans, despite the uterine and placental differences between these two species. With the exception of slightly earlier growth deceleration, curves for head and long-bone growth resembled those for singleton ovine fetuses. Ovine fetal growth patterns (like those of humans) in singleton and twin pregnancies are similar.

Abdomen↗
PubMed2001

PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells.

The p85-associated phosphatidylinositol (PI) 3-kinase/Akt pathway mediates the oestradiol-induced S-phase entry and cyclin D1 promoter activity in MCF-7 cells. Experiments with Src, p85alpha and Akt dominant-negative forms indicate that in oestradiol-treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3-kinase and Akt activities in MCF-7 cells. In NIH 3T3 cells expressing ERalpha, a dominant-negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3-kinase activities in MCF-7 cells. In turn, stimulation of Src activity is abolished in ERalpha-expressing NIH 3T3 fibroblasts by co-transfection of the dominant-negative p85alpha and in MCF-7 cells by the PI3-kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross-talk between PI3-kinase and Src. Hormone stimulation of MCF-7 cells rapidly triggers association of ERalpha with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src- and PI3-kinase-dependent pathways, which converge on cell cycle progression.

Breast Neoplasms↗
PubMed2001

cAMP signaling selectively influences Ras effectors pathways.

Thyrotropin (TSH) stimulates survival and growth of thyroid cells via a seven transmembrane G protein-coupled receptor. TSH elevates the intracellular cyclic AMP (cAMP) levels activating protein kinase A (PKA). Recent evidence indicates that p21 Ras is required for TSH-induced mitogenesis, but the molecular mechanism(s) is not known. Here we report that Ras p21 activity is necessary for the Go- G1 transition in TSH induced cycle and that the downstream effector of Ras upon TSH signaling is p85-p110 PI3K. We show that PI3K inhibitors block TSH-induced DNA synthesis, cAMP-PKA stimulate the formation of the complex PI3K-p21 Ras and reduce the complex Ras-Raf1 in thyroid and other cells types. Moreover, PKA phosphorylates immunoprecipitated p85 and PKA phosphorylation of cell extracts significantly stimulates the formation of the complex PI3K-Ras. We suggest that PKA phosphorylates p85 and stabilizes the complex p110-p85, enhancing the interaction PI3K and p21 Ras. Simultaneously, cAMP inhibits Raf-1-ERK signaling by decreasing Raf1 availability to Ras. Under these circumstances PI3K signaling is favored. These results indicate that PI3K is an important mediator of Ras effects in cAMP-induced proliferation and illustrates how cAMP can selectively influence Ras effector pathways.

Cell Cycle↗
PubMed2001

A factor analysis of signs and symptoms of the manic episode with Bech-Rafaelsen Mania and Melancholia Scales.

BACKGROUND: Several factor analyses of signs and symptoms of mania have been reported using different rating scales. We propose here that the use of two instruments well known in the European literature may be useful in detecting the structure of manic episodes. METHOD: We investigated the pattern of symptoms in a group of 124 bipolar inpatients hospitalised for a manic episode. We conducted a factor analysis of the broad range of psychiatric symptoms covered by the Bech-Rafaelsen Mania Scale (BRMaS) and Melancholia Scale (BRMeS). RESULTS: Five eigen values were greater than unity, which determined the number of factors computed. The five factors captured 66.7% of the total variance. Following rotation, five factors were clinically relevant. CONCLUSION: This suggests that both euphoric activation and depression are prominent in this sample.

Bipolar Disorder↗
PubMed2000

Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation.

Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor beta with Src, activates the Src/Raf-1/Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar findings for oestradiol receptor alpha were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK(-) abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor alpha or beta is detected using glutathione S:-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor alpha and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor alpha with Src and consequent activation of Src in intact Cos cells.

Androgen Antagonists↗
PubMed2000

Cognitive symptoms of mania in pure and mixed episodes evaluated with the Positive and Negative Syndrome Scale.

To investigate the clinical specificity of mixed affective patients, we compared the clinical characteristics of pure manic patients with those of mixed manic patients. The clinical symptoms of 146 bipolar inpatients hospitalized for a manic episode were assessed by means of the Positive and Negative Syndrome Scale. Mixed patients showed more positive and cognitive symptoms and among these the lack of judgement and insight was prominent. Further studies are needed to clarify the specificity of lack of insight of the mixed bipolar patients.

Adult↗
PubMed1999

Non-transcriptional action of oestradiol and progestin triggers DNA synthesis.

The recent findings that oestradiol and progestins activate the Src/Ras/Erks signalling pathway raise the question of the role of this stimulation. Microinjection experiments of human mammary cancer-derived cells (MCF-7 and T47D) with cDNA of catalytically inactive Src or anti-Ras antibody prove that Src and Ras are required for oestradiol and progestin-dependent progression of cells through the cell cycle. The antitumoral ansamycin antibiotic, geldanamycin, disrupts the steroid-induced Ras-Raf-1 association and prevents Raf-1 activation and steroid-induced DNA synthesis. Furthermore, the selective MEK 1 inhibitor, PD 98059, inhibits oestradiol and progestin stimulation of Erk-2 and the steroid-dependent S-phase entry. The MDA-MB231 cells, which do not express oestradiol receptor, fail to respond to oestradiol in terms of Erk-2 activation and S-phase entry. Fibroblasts are made equally oestradiol-responsive in terms of DNA synthesis by transient transfection with either the wild-type or the transcriptionally inactive mutant oestradiol receptor (HE241G). Co-transfection of catalytically inactive Src as well as treatment with PD98059 inhibit the oestradiol-dependent S-phase entry of fibroblasts expressing either the wild-type oestrogen receptor or its transcriptionally inactive mutant. The data presented support the view that non-transcriptional action of the two steroids plays a major role in cell cycle progression.

3T3 Cells↗
PubMed1999

[The antioxidants (coenzyme Q10) in materno-fetal physiopathology].

BACKGROUND: Coenzyme Q10 (ubidecarenone) is a torpeniod molecule mainly located in bacterial and mitochondrial membranes. It is a part of a specific enzyme system and acts primarily on the transport of electrons in the mitochondrial respiratory chain. It performs an antioxidant action. We studied both fetal and maternal coenzyme Q10 plasma levels in physiological conditions and also in the presence of some pathologies. METHODS: As regards the maternal side, we selected 483 pregnancies, performing 615 blood samples, and divided them into four groups: A: physiological pregnancies; B: spontaneous abortions; C: threatened abortions; D: threatened late abortions and threatened pre-term deliveries. We then selected 61 pregnancies which differed from the previous ones and determined Q10 levels in fetal samples obtained by cordocentesis. We divided a control group from pathological groups: intra-uterine growth retardation (IUGR); Rh-isoimmunization (with intra-uterine transfusion), non immune fetal hydrops, fetal malformations. Coenzyme Q10 levels were determined in only one laboratory by high-performance liquid chromatography (HPLC). Coenzyme Q10 concentrations were expressed as mg/ml, the data as the mean + SD. Statistical analysis was performed employing a linear regression model and the student "t"-test. RESULTS: After working out a normality curve of CoQ10 levels in maternal blood, we noticed that the levels of Coenzyme Q10 were low in spontaneous abortions, in threatened late abortions and in threatened pre-term deliveries. We determined the value of 0.3 mg/ml as a cutoff to differentiate the fetal from the adult values. Moreover, CoQ10 values turned out to be increased only in fetuses affected by non-immune fetal hydrops. CONCLUSIONS: Accordingly, we can say that maternal CoQ10 plasma levels can be considered as a marker of pathological uterine contractile activity. There is a substantial increase in CoQ10 fetal plasma levels in fetuses affected by hypoxic hypoxia and also in those affected by non-immune fetal hydrops.

Abortion, Spontaneous↗
PubMed1998

Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor.

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21(ras)/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21(ras)/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

Animals↗
PubMed1996

Estradiol activation of human colon carcinoma-derived Caco-2 cell growth.

This is the first report on estrogen-dependent growth of human-derived colon carcinoma cells. Under selected conditions, growth of subconfluent Caco-2 cells is triggered by estradiol. Cell growth is estradiol concentration dependent, with maximal effect occurring at about 0.4 nM. Growth is prevented by two different antiestrogens: the partial agonist, OH-Tamoxifen, and the pore antagonist, ICI 182,780. The growth effect is specific for estradiol since other hormonal steroids tested do not affect cell growth. The amount of estradiol receptor in subconfluent Caco-2 cells, detected by blot with monoclonal antibodies directed against the receptor as well as estradiol binding assays, is similar to that of the classical estradiol-responsive, human mammary cancer-derived MCF-7 cells. Estradiol treatment of subconfluent Caco-2 cells rapidly and reversibly stimulates four important intermediates in a signal transduction pathway that is known to trigger cell proliferation: two members of the large family of c-src-related tyrosine kinases, c-src and c-yes, and two serine/threonine kinases, the mitogen-activated protein (MAP) kinases, erk-1 and erk-2. Tyrosine kinases activated by estradiol are up-stream MAP kinases and Caco-2 cell proliferation. In fact, genistein, a specific tyrosine kinase inhibitor, abolishes the estradiol stimulatory effect on both erk-2 activity and cell proliferation. Our findings show that in subconfluent Caco-2 cells, the estradiol-receptor complex activates the c-src, c-yes/MAP kinase pathway and activates growth. This could have important implications for the understanding of human intestinal carcinogenesis.

Caco-2 Cells↗
PubMed1996

Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor.

Breast Neoplasms↗