S Sestini

Università degli Studi di Siena, Siena, Tuscany, Italy

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Publications (44)74.85 Total impact

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    ABSTRACT: Macrophage Migration Inhibitory Factor (MIF) is a pivotal regulator of innate and acquired immunity affecting the response and behavior of macrophages and lymphocytes. However, a number of studies indicated wider physiological functions for this cytokine to include key-roles in reproductive biology. The present study was designed to clone the coding sequence of sheep MIF, to examine the characteristics of the protein in vitro, and to evaluate its expression in sheep tissues and in the ewe reproductive tract in vivo. Ovine MIF cDNA consisted of 348 nucleotides encoding a 115 amino acids protein with an estimated molecular mass of 12,343 Da and an isoelectric point of 7.68. Sheep MIF shared high amino acid identity with the other mammalian MIF family members and showed parallel functions to human MIF, displaying enzymatic oxoreductase activity and inducing monocyte transmigration. Expression studies detected a MIF transcript in all the sheep tissues examined. Among reproductive tissues, MIF mRNA and protein were detected in the ovary, oviduct, uterus and placenta. These results indicate that sheep MIF shares crucial features with other MIF family members and delineate its potential involvement in several aspects of ovine physiology.
    Cytokine 03/2011; 54(3):315-23. · 2.52 Impact Factor
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    ABSTRACT: The enzyme poly(ADP-ribose) polymerase (PARP-1, EC 2.4.2.30) is activated by DNA strand breaks caused by several agents and utilizes NAD to form polyADPR, bound to acceptor proteins. The involvement of PARP-1 in autoimmune diseases has been suggested: antiPARP autoantibodies are described in systemic lupus erythematosus (SLE), DNA strand breaks have been evidenced in systemic sclerosis (SSc). We tested poly(ADP-ribosyl)ation activity and NAD concentration in PMC from patients affected by SLE or SSc and from controls. Lower PARP-1 activity and higher NAD concentration were observed in pathological conditions than controls, supporting the role of PARP-1 activation in modulating NAD concentration.
    Nucleosides Nucleotides &amp Nucleic Acids 06/2010; 29(4-6):471-5. · 0.71 Impact Factor
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    ABSTRACT: The aim of this study is to investigate the role of poly(ADP-ribose) polymerase (PARP), involved in DNA repair and in autoimmune pathologic conditions such as systemic lupus erythematosus (SLE) and both limited systemic sclerosis (lSSc) and diffuse systemic sclerosis (dSSc), to assess its possible implication in the pathogenetic processes. The relationship between PARP activity and the intracellular concentration of its substrate nicotinamide adenine dinucleotide (NAD) is also investigated. Peripheral mononuclear cells (PMC) from controls and patients with SLE, lSSc, and dSSc were irradiated with ultraviolet light (UV) and PARP activity was assayed by a radiochemical method. Pyridine nucleotide concentrations were assayed by a high-performance liquid chromatography-linked method. PARP activity was detectable in nonirradiated cells and showed similar values in all groups. The activity significantly increased after UV irradiation in control, SLE, and lSSc cells, but not in dSSc cells. Irradiated PMC from both SLE and dSSc showed lower enzyme activity with respect to irradiated controls. Higher intracellular NAD content was found in all of the pathologic conditions in comparison to values in the control; this difference was statistically significant in dSSc. Our data demonstrate a lower PARP activity in response to UV damage in PMC from patients affected by the above pathologic conditions compared with controls. An inverse relationship between PARP activity and NAD content was also observed.
    Human immunology 05/2009; 70(7):487-91. · 2.55 Impact Factor
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    ABSTRACT: The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT(-) and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT(-) mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.
    Metabolic Brain Disease 04/2009; 24(2):311-9. · 2.33 Impact Factor
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    ABSTRACT: The long-term effects of orthodontic appliances in the oral environment and the subsequent leaching of metals are relatively unknown. A method for determining the effects of various types of soldering and welding, both of which in turn could lead to leaching of metal ions, on the growth of osteoblasts, fibroblasts, and oral keratinocytes in vitro, is proposed. The effects of cell behaviour of metal wires on osteoblast differentiation, expressed by alkaline phosphatase (ALP) activity; on fibroblast proliferation, assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenil)-2H-tetrazolium-phenazine ethosulphate method; and on keratinocyte viability and migration on the wires, observed by scanning electron microscopy (SEM), were tested. Two types of commercially available wires normally used for orthodontic appliances, with a similar chemical composition (iron, carbon, silicon, chromium, molybdenum, phosphorus, sulphur, vanadium, and nitrogen) but differing in nickel and manganese content, were examined, as well as the joints obtained by electrical resistance welding, traditional soldering, and laser welding. Nickel and chromium, known as possible toxic metals, were also examined using pure nickel- and chromium-plated titanium wires. Segments of each wire, cut into different lengths, were added to each well in which the cells were grown to confluence. The high nickel and chromium content of orthodontic wires damaged both osteoblasts and fibroblasts, but did not affect keratinocytes. Chromium strongly affected fibroblast growth. The joint produced by electrical resistance welding was well tolerated by both osteoblasts and fibroblasts, whereas traditional soldering caused a significant (P < 0.05) decrease in both osteoblast ALP activity and fibroblast viability, and prevented the growth of keratinocytes in vitro. Laser welding was the only joining process well tolerated by all tested cells.
    The European Journal of Orthodontics 01/2007; 28(6):567-72. · 1.08 Impact Factor
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    ABSTRACT: Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.
    Biochimica et Biophysica Acta 02/2006; 1762(1):29-33. · 4.66 Impact Factor
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    ABSTRACT: Aim of this study was to ascertain whether allopurinol, usually administered to hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient patients, or metabolites abnormally increased in HPRT deficient erythrocytes (NAD, PPribP) could be directly responsible for the reported increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NADsynthetase (NADs) in these patients. No direct effect of the mentioned metabolites was demonstrated.
    Nucleosides Nucleotides &amp Nucleic Acids 11/2004; 23(8-9):1189-91. · 0.71 Impact Factor
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    ABSTRACT: Biocompatibility of metals for dental use was tested using a three-dimensional model consisting of oral keratinocytes cultured on de-epidermised sub-mucosa. The toxicity of orthodontic metallic wire and soldering material was assessed through parameters such as the morphology and growth rate of the keratinocytes, as well as by classical histology, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The sharp composition of metallic wires and soldering materials was assessed by Auger Electron Spectroscopy (AES). The results of our experiment showed that the new model revealed inhibition of keratinocyte growth and stratification near soldering material, whereas mucosal cells were able to grow and layer out on dental wire. It is concluded that this experimental model, which simulates the oral environment, is useful for studying the effects of materials for dental use for its sensibility and reproducibility. Moreover it can provide morpho-functional information which cannot be achieved by traditional methods.
    Bulletin du Groupement international pour la recherche scientifique en stomatologie & odontologie 01/2004; 46(2-3):63-71.
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    ABSTRACT: Pathologies associated with rare inherited disorders affecting purine metabolic pathways range from renal failure to neurological dysfunction and immunodeficiency. The disorders are usually diagnosed by measuring enzyme activities in hemolysates. A non-radiochemical HPLC-linked method is described for simultaneous determination of the activities of hypoxanthine-guanine phosphoribosyltransferase (HPRT: E.2.4.2.8.), adenine phosphoribosyltransferase (APRT: E.2.4.2.7.), adenosine deaminase (ADA: E.3.5.4.4.) and purine nucleoside phosphorylase (PNP: E.2.4.2.1.) in dried blood spots. 7-mm-diameter blood spots stored at 4 degrees C or room temperature were transferred to an Eppendorf tube and eluted with 500-microl 0.1 mol/l Tris-HCl buffer, pH 7.4. The eluate was added to substrate solutions and incubated at 37 degrees C. Reaction products were analysed by HPLC. The enzyme activities tested in spot eluates were similar to those in erythrocyte lysates from the same subjects. None of the enzymatic activities tested were significantly affected by different storage temperatures. The main advantages of the proposed method are small blood volume required, easy sample collection and transfer, and accurate results. The method is therefore suitable for screening inborn errors of purine metabolism even in newborns.
    Clinica Chimica Acta 11/2002; 324(1-2):135-9. · 2.85 Impact Factor
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    ABSTRACT: Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.
    Biochimica et Biophysica Acta 06/2002; 1587(1):45-52. · 4.66 Impact Factor
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    ABSTRACT: Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.
    Journal of Cellular Biochemistry 02/2001; 80(3):360-6. · 3.06 Impact Factor
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    ABSTRACT: Pyridine nucleotide levels and the activities of enzymes involved in NAD synthesis (nicotinic acid phosphoribosyltransferase, nicotinic acid- and nicotinamide mononucleotide-adenylyltransferase) have been assayed in human normal lymphocytes by an HPLC method using radioactive or nonradioactive substrates. NAD concentration was 46.4 +/- 17.2 pmol 10(-6) cells, and that of NADP was 14.5 +/- 3.9 pmol 10(-6) cells (mean +/- standard deviation). The adenylyltransferase activity using nicotinic acid mononucleotide as substrate was 1.530 +/- 0.216 nmol h(-1) 10(-6) cells, using nicotinamide mononucleotide was 1.466 +/- 0.354 nmol h(-1) 10(-6) cells. The apparent K(M) values were 0.015 mM for the former substrate and 0.167 mM for the latter. The mean activity of nicotinic acid phosphoribosyltransferase was 0.038 +/- 0.014 nmol h(-1) 10(-6) cells, and the apparent K(M) for nicotinic acid was 0.165 mM. The proposed methods, easy and rapid to perform, are reliable and sensitive, avoiding the use of radiolabels except for NAPRT and displaying a very low activity. The reported findings, together with the previous ones in human erythrocytes, can provide an useful base to investigate NAD metabolism in humans through the study of blood cells.
    Archives of Biochemistry and Biophysics 08/2000; 379(2):277-82. · 3.37 Impact Factor
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    ABSTRACT: This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in T-47D cells, while 11beta-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11beta-HSD catalytic activity was elevated 11-fold, while estrone (E(1)), estradiol (E(2)) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11beta-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11beta-HSD2 gene expression, increasing the steady-state levels of 11beta-HSD2 mRNA. Taken together, these results demonstrate that 11beta-HSD2 is the 11beta-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.
    The Journal of Steroid Biochemistry and Molecular Biology 05/2000; 72(5):239-47. · 3.98 Impact Factor
  • F Arcuri, S Sestini, M Cintorino
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    ABSTRACT: Glucocorticoid hormone action in target tissues is modulated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which interconverts active cortisol and corticosterone and their inert 11-keto metabolites, cortisone and 11-dehydrocorticosterone. Two different 11 beta-HSD isoforms exist: a low-affinity NADP-dependent dehydrogenase/oxoreductase (11 beta-HSD1) and a high-affinity NAD-dependent dehydrogenase (11 beta-HSD2). This brief review describes the expression and distribution of 11 beta-HSD isoforms in human placenta. In particular, it discusses the results of studies dealing with the expression of 11 beta-HSD activity in experimental models representative of the fetomaternal interface in the early gestation. The findings have implications in terms of protection of the fetus against corticosteroid toxicity and modulation of active glucocorticoid levels and their biological effects in early pregnancy.
    Seminars in reproductive endocrinology 02/1999; 17(1):53-61.
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    ABSTRACT: Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.
    Life Sciences 02/1999; 64(26):2479-87. · 2.56 Impact Factor
  • European Journal of Cancer 09/1998; 34:84-84. · 5.06 Impact Factor
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    ABSTRACT: This study evaluated the levels and the enzymatic characteristics of 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) of chorionic villi isolated from first trimester human placenta. The results demonstrated a predominant expression of the NAD-dependent dehydrogenase isoform (11beta-HSD2) over the NADP-dependent oxoreductase (11beta-HSD1). Thus, in tissue homogenates exogenous NAD increased the conversion of corticosterone to 11-dehydrocorticosterone of about 14-fold while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in villous tissue while 11beta-HSD1 mRNA levels were undetectable. In addition, immunohistochemical staining localized the 11beta-HSD2 protein to syncytiotrophoblasts and cell columns of the chorionic villi. These results suggest roles for the trophoblast-associated 11beta-HSD2 oxidative activity in modulating the exposure of the embryo to active glucocorticoids in the early gestation and in regulating trophoblasts invasion of the uterine wall.
    Molecular and Cellular Endocrinology 07/1998; 141(1-2):13-20. · 4.04 Impact Factor
  • Advances in experimental medicine and biology 02/1998; 431:181-4. · 1.83 Impact Factor
  • European Journal of Cancer - EUR J CANCER. 01/1998; 34.
  • V Micheli, S Sestini
    Methods in Enzymology 02/1997; 280:211-21. · 2.00 Impact Factor