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V Sica

Publications and source records attributed to V Sica.

At least 19 recordsLinked to original sources
PubMed2003

Microarray analysis: a novel research tool for cardiovascular scientists and physicians.

The massive increase in information on the human DNA sequence and the development of new technologies will have a profound impact on the diagnosis and treatment of cardiovascular diseases. The microarray is a micro-hybridisation based assay. The filter, called microchip or chip, is a special kind of membrane in which are spotted several thousands of oligonucleotides of cDNA fragments coding for known genes or expressed sequence tags. The resulting hybridisation signal on the chip is analysed by a fluorescent scanner and processed with a software package utilising the information on the oligonucleotide or cDNA map of the chip to generate a list of relative gene expression. Microarray technology can be used for many different purposes, most prominently to measure differential gene expression, variations in gene sequence (by analysing the genome of mutant phenotypes), or more recently, the entire binding site for transcription factors. Measurements of gene expression have the advantage of providing all available sequence information for any given experimental design and data interpretation in pursuit of biological understanding. This research tool will contribute to radically changing our understanding of cardiovascular diseases.

Cardiac Output, Low↗
PubMed2001

Effects of nitric oxide-releasing aspirin versus aspirin on restenosis in hypercholesterolemic mice.

Restenosis is due to neointimal hyperplasia, which occurs in the coronary artery after percutaneous transluminal coronary angioplasty (PTCA). During restenosis, an impairment of nitric oxide (NO)-dependent pathways may occur. Concomitant hypercholesterolemia may exacerbate restenosis in patients undergoing PTCA. Here, we show that a NO-releasing aspirin derivative (NCX-4016) reduces the degree of restenosis after balloon angioplasty in low-density lipoprotein receptor-deficient mice and this effect is associated with reduced vascular smooth muscle cell (VSMC) proliferation and macrophage deposition at the site of injury. Drugs were administered following both therapeutic or preventive protocols. We demonstrate that NCX-4016 is effective both in prevention and treatment of restenosis in the presence of hypercholesterolemia. These data indicate that impairment of NO-dependent mechanisms may be involved in the development of restenosis in hypercholesterolemic mice. Although experimental models of restenosis may not reflect restenosis in humans in all details, we suggest that a NO-releasing aspirin derivative could be an effective drug in reducing restenosis following PTCA, especially in the presence of hypercholesterolemia and/or gastrointestinal damage.

Animals↗
PubMed1996

17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.

MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.

Breast Neoplasms↗
PubMed1996

17 beta-Estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation.

HMG-CoA reductase inhibitors, such as Lovastatin and Simvastatin, cause cell cycle arrest by interfering with the mitogenic activity of mitogens present in culture media. Cells are induced to pause in G1 and can readily resume growth upon removal of the enzymatic block. Estrogens, acting via their nuclear receptor, are mitogens for different normal and transformed cell types, where they foster cell cycle progression and cell division. In estrogen-responsive MCF-7 human breast cancer cells, but not in non responsive cells, 17 beta-estradiol (E2) induces cells arrested with Lovastatin or Simvastatin to proliferate in the presence of inhibitor, without restoring HMG-CoA reductase activity or affecting the protein prenylation pattern. Mitogenic stimulation of G1-arrested MCF-7 cells with E2 includes primary transcriptional activation of c-fos, accompanied by transient binding in vivo of the estrogen receptor and/or other factors to the ERE and the estrogen-responsive DNA region of this proto-oncogene, as detected by dimethylsulphate genomic footprinting analysis. Mitogenic stimulation of growth-arrested MCF-7 cells by E2 occurs, under these conditions, without evident activation of ERK-1 and -2 kinases, and thus independently from the mitogen-responsive signal transduction pathways that converge on these enzymes.

Base Sequence↗
PubMed1995

In vivo functional analysis of the mouse estrogen receptor gene promoter: a transgenic mouse model to study tissue-specific and developmental regulation of estrogen receptor gene transcription.

Understanding the molecular and morphological basis of estrogen responsiveness in the various tissues and organs that make up an adult organism and its onset during ontogenesis requires identification of the genetic controls that determine timed expression of the estrogen receptor (ER) gene in multiple cell types. With this goal in mind, we describe here the results of the functional analysis of the mouse (m) ER gene promoter, carried out in vivo in transgenic mice. The mER gene promoter was cloned and spliced to the coding sequence of the bacterial lacZ gene (fused to the nuclear localization signal of SV40 large T: nls-beta-GAL) and then stably reintegrated into the genome of mice. Analysis of beta-GAL mRNA and protein expression in multiple organs of both female and male transgenic animals was then performed. Results show that the transgenic mER promoter, much like the endogenous one, is active in several organs and tissues of adult female and male mice. The first 0.4 kilobases of 5'-flanking DNA (up to -364) are sufficient to direct widespread expression of the transgene in mouse organs. This indicates that genetic elements functional in various cell types are included in this segment. Furthermore, the first exon and intron of the mER gene are necessary to achieve sexually dimorphic expression of the transgene in neurons located at specific sites within the central nervous system. These mER promoter transgenic mice will be useful in mapping estrogen- responsive cell types under different physiological and pathological conditions in vivo, in defining ontogenesis of estrogen action in the mouse, and in studying the mechanisms that regulate ER gene transcription.

Animals↗
PubMed1994

Endemic goiter and thyroid function in central-southern Sardinia. Report on an extensive epidemiological survey.

UNLABELLED: Although the existence of endemic goiter and cretinism in Sardinia is known since to ancient time, scanty information collected according to WHO criteria is available. In the present paper the results of an extensive epidemiological survey carried out in juvenile population living in some rural and/or hilly villages in the provinces of Nuoro and Oristano in Central-Southern Sardinia and in urban area of Cagliari, are reported. In the majority of the villages the mean urinary iodine excretion was lower than 60 micrograms/L; the goiter prevalence ranged between 39% and 61% in the district of Nuoro and between 21% and 56% in the district of Oristano. In the control area the urinary iodine excretion was 105 micrograms/L with a goiter prevalence of 12%. Goiter prevalence was not always inversely related to urinary iodine excretion. No relevant thyroid function alterations were found. IN CONCLUSION: 1) in extraurban areas of Central-Southern Sardinia mild to moderate iodine deficiency and endemic goiter are still a widespread problems; 2) also in urban area endemic goiter prevalence is still higher than 10%; 3) extemporary urinary samples are inadequate for assessing the severity of goiter endemia in mild to moderate iodine deficiency; 4) in mildly affected districts palpation is inaccurate for assessing the prevalence of goiter; 5) no relevant alterations of thyroid function were documented in juvenile population.

Adolescent↗
PubMed1993

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

Animals↗
PubMed1993

Identification of a specific pattern of "immediate-early" gene activation induced by estrogen during mitogenic stimulation of rat uterine cells.

Estrogen hormones are potent mitogens for certain target tissues, where they stimulate cell growth by inducing recruitment of quiescent cells in cycle and by fostering cell cycle progression. To define the molecular bases of the mitogenic action of these steroid hormones, the pattern of "immediate-early" gene expression was monitored during the early phases of estrogen stimulation of rat uterine cells in vivo. Nuclear run-on transcription and/or Northern blot RNA analysis indicate that c-jun, junB, jun-D, c-fos, TIS 1 (also called NGFI-B or nur/77) and TIS 8 (zif-268, krox24, egr-1, or NGFI-A) genes are all transiently activated in the uterus (up to 20-fold) within 30-120 min after treatment of adult ovariectomized rats with a mitogenic dose of 17b-estradiol. Conversely, JE gene mRNA accumulates progressively in estrogen-stimulated uterine cells, whereas TIS 11 and 21 genes are only slightly responsive to the hormone (less than twofold induction) and fos B,fra-1,fra-2,krox20 (egr-2), TIS 7 and 10, KC, and c-rel mRNAs are undetectable in rat uterus either before or after estrogen treatment. Stimulation in the presence of cycloheximide shows that only c-jun, jun-D, c-fos, and JE gene activations are primary responses to the hormone in rat uterine cells. These findings establish the direct mitogenic role of estrogen and identify for the first time a specific genetic program activated by these steroid hormones during stimulation of target cell proliferation. Furthermore, since most of the activated genes encode for transcription factors, these results enable us to envision how the mitogenic signal transmitted by the hormone can be elaborated and amplified within target cells by the products of estrogen-responsive genes, leading to a cascade of growth-dependent gene regulation, cell cycle progression, and, ultimately, cell division.

Animals↗
PubMed1992

Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells.

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.

Actins↗
PubMed1991

Therapy of Graves' disease with sodium ipodate is associated with a high recurrence rate of hyperthyroidism.

To evaluate the long-term efficacy of sodium ipodate (IPO) in the treatment of hyperthyroid Graves' disease, we studied 12 consecutive patients with Graves' hyperthyroidism treated only with 500 mg IPO po daily for several weeks to 22 months. Serum thyroid hormone concentrations markedly decreased and serum free T3 values normalized in all patients within 7 days of therapy. Five patients (42%, Group 1) were euthyroid after 6 weeks of IPO treatment and remained so until IPO was discontinued after 22 months. Recurrence of hyperthyroidism after drug withdrawal occurred in only one of these Group 1 patients, who was promptly responsive to a second course of IPO. In contrast, seven of 12 patients (58%, Group 2) relapsed with recurrent hyperthyroidism between 14 and 42 days of IPO therapy. After IPO was withdrawn, these Group 2 patients were treated with methimazole (20-30 mg/day, initial dose), but the therapeutic response was poor and delayed. Two patients were still hyperthyroid after 6 months of methimazole treatment. Elevated serum FT3 concentrations were observed in the Group 2 patients at 21 days following the early normalization of serum FT3 concentrations. No changes in serum thyroglobulin and thyroid microsomal and TSH-receptor autoantibody titers were observed in either groups during IPO therapy. In conclusion, the results of the present study demonstrate that IPO rapidly restores euthyroidism, but its prolonged administration is associated with a high rate of relapse of hyperthyroidism and a poor response to subsequent methimazole treatment and that long-term IPO administration does not affect humoral markers of thyroid autoimmunity.

Adolescent↗
PubMed1990

Activation of 'immediate-early' genes by estrogen is not sufficient to achieve stimulation of DNA synthesis in rat uterus.

17 beta-estradiol, a long acting estrogen that is mitogenic for rat uterus in vivo, or the short acting estrogens estriol and 16 alpha-estradiol, not mitogenic on their own, were injected into adult, castrated rats and their effect on uterine gene expression and rate of DNA synthesis were compared. All three compounds increased steady-state mRNA concentration of c-fos, c-jun and c-myc proto-oncogenes to comparable levels (2 hrs after treatment), whereas only 17 beta-estradiol was found to stimulate significantly DNA synthesis (20-22 hrs later). Based on the different retention time of the tested estrogens in rat tissues, it is concluded that a short exposure to the hormone is sufficient to render uterine cells competent to progress through the cell cycle, via activation of 'immediate-early' genes expression, but that stimulation of DNA synthesis requires further changes, achieved via a prolonged exposure of the cells to the estrogenic stimulus.

Animals↗
PubMed1989

Purification to homogeneity of the major "4S" PAH binding protein from "non responsive" DBA/2N mouse liver by affinity chromatography.

DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.

Animals↗
PubMed1989

"4S" polycyclic aromatic hydrocarbon binding protein. Its role as a benzo(a) pyrene cytosolic carrier to the microsomes of DBA/2N mouse liver.

Rodent liver cytosol and other biological systems contain two proteins that bind polycyclic aromatic hydrocarbons (PAH) in a non covalent manner and that sediment at a different rate when centrifuged on sucrose gradient. The role of the smaller protein ("4S" PAH-BP) was studied. When DBA/2N mouse liver homogenate was incubated with 3H-BaP, most of the radioactivity was found in the microsomal subcellular fraction. The cytosol binding activity apparently decreased but reincubation of the cytosol with the radioactive ligand completely restored "4S" PAH-BP activity. The microsomal uptake of 3H-BaP can be studied in a reconstituted system in which microsomes are incubated with radioactive benzo(a)pyrene in the presence of crude cytosol. In these conditions the microsomal uptake rate of 3H-BaP increased with the temperature and at 37 degrees C ten minutes were required to reach the plateau. When cytosol was substituted by HEDG buffer, the amount of radioactivity found in the microsomes decreased drastically. 0.2 microM was the benzo(a)pyrene concentration required to saturate the microsomes. When microsomes were incubated with ammonium sulfate cytosolic fractions or with homogeneously purified "4S" PAH-BP, the 3H-BaP uptake was restored and reached the maximum with 3 micrograms/ml of purified protein. The radioactive benzo(a)pyrene bound to microsomes was oxidated in the presence of NADPH regenerating system. The oxidated products were discharged from microsomes only when "4S" PAH-BP was either present during the incubation or added at its end. Thus, this protein is able to transfer benzo(a)pyrene to the microsomal metabolization sites and to facilitate the release of oxidized products and, presumably, bind them.

Ammonium Sulfate↗
PubMed1988

Absence of serum thyroid hormone autoantibodies in patients chronically treated with amiodarone.

The role of iodine in the pathogenesis of thyroid hormone autoantibodies (THAA) was evaluated in a large series (n = 223) of patients submitted to chronic treatment (3-36 months) with the iodine-rich drug, amiodarone. Positive anti-T3 autoantibody (AbT3) tests were found only in one patient, whereas tests for anti-T4 autoantibody (AbT4) were negative in all cases. Likewise, the incidence of THAA in the control groups of patients with spontaneous thyroid disorders was low. The overall prevalence of THAA in the present series of 803 patients was 1.2% for AbT3 and 0.1% for AbT4. The present data strongly suggest that iodine plays a minor role, if any, in the occurrence of THAA.

Amiodarone↗
PubMed1988

The effect of flutamide on basal and ACTH-stimulated plasma levels of adrenal androgens in patients with advanced prostate cancer.

The effect of flutamide on basal and ACTH-stimulated plasma levels of adrenal androgens was investigated in 6 patients with untreated advanced prostate cancer, aged 52-75 yr. Flutamide was administered (250 mg three times daily) for 10 days; before and after treatment, a synthetic ACTH1-24 stimulation test (250 micrograms im, with blood sampling immediately before and 60 min after the stimulus) was performed. Basal plasma 17OH-pregnenolone (delta 5-17OHP), 170H-progesterone (delta 4-17OHP), androstenedione (A), dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) were unchanged by flutamide treatment. In contrast, basal plasma testosterone (T) concentrations significantly increased (p less than 0.05). The response of cortisol delta 4-17OHP, delta 5-17OHP, A and DHEA to ACTH, as well as the ACTH-stimulated delta 5-17OHP/delta 4-17OHP, delta 5-17OHP/DHEA, delta 4-17OHP/A and DHEA/A ratios, were unchanged by flutamide treatment. These findings indicate that: a) Short-term flutamide administration enhances testicular steroidogenesis, via augmented LH pulse frequency; b) Adrenal steroidogenesis seems to be not affected by the drug, since ACTH-stimulated plasma levels of adrenal androgens and precursors/products ratios were unchanged.

Adrenal Glands↗
PubMed1987

"4 S" polycyclic aromatic hydrocarbon binding protein: further characterization and kinetic properties.

A protein that binds polycyclic aromatic hydrocarbons (PAHs) with high affinity and sediments in a sucrose gradient at 4 S has been described in rat liver cytosol. This "4 S" PAH binding protein precipitates at a 40-60% ammonium sulfate saturation. This partial purification procedure allows assay of this protein by using purified 3H-benzo(a)pyrene (3H-BaP) as radioactive ligand and dextran-coated charcoal as adsorbent for unreacted 3H-BaP. The 3H-BaP binding activity measured as a function of pH shows its maximum activity between pH 7.3 and 10.5. The "4 S" PAH binding protein is stable up to 42 degrees C even in the absence of the ligand. At 65 degrees C the binding sites for 3H-BaP are destroyed. The binding activity assayed as a function of protein concentration is linear between 0.4 and 2 mg/ml at 0 degrees C, whereas at 37 degrees C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine X HCl (3 M) and urea (5 M) for 20 min at 4 degrees C inhibits the PAH binding completely to the "4 S" protein. Quick dilution or dialysis does not restore the binding activity. The dissociation rate of the "4 S" PAH binding protein measured in the presence of an excess of unlabeled ligand at 0 degrees C is biphasic and shows a two-step, first-order kinetic pattern. At 37 degrees C the dissociation rate is linear and faster, and is complete after 5 min of incubation. The association rate shows the same behavior: the binding is complete after 10 min at 0 degrees C, whereas at 37 degrees C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 degrees C and 37 degrees C are respectively 2.45 X 10(-9) M and 1.09 X 10(-9) M. The high rates of association and dissociation of BaP to "4 S" PAH binding protein were used to set up an assay to exchange radioactive 3H-BaP with cold BaP.

Animals↗