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Biomedical subjects

F Moretti

Publications and source records attributed to F Moretti.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of exogenous p53 transduction in thyroid tumor cells with different p53 status.

Recovery of p53 function in undifferentiated thyroid carcinoma cells carrying an altered p53 gene is able to modify cell tumorigenic properties. It is not known whether such an effect may also be achieved in thyroid cancer cells expressing wild-type p53, as in the majority of differentiated thyroid carcinomas. Effects of p53 transduction in a thyroid carcinoma cell line (FRO) exhibiting a wild-type endogenous p53 gene, in comparison to a cell line (WRO) exhibiting mutant p53, were investigated by using an inducible chimeric construct containing human p53 complementary DNA fused to the ligand binding domain of the estrogen receptor (p53ER). FRO cells were unaffected by exogenous p53 expression in terms of both proliferation and viability. On the contrary, p53 reexpression in WRO cells containing hemizygous mutated p53 allele caused a strong growth inhibition due to cell accumulation in the G1 phase of the cell cycle. In addition, exogenous p53 did not influence FRO cell behavior in response to TSH treatment or modify cell resistance to the chemotherapeutic agent, doxorubicin. Our results indicate that exogenous expression of wild-type p53 affects thyroid tumorigenic properties only in cells carrying an altered p53, whereas it is ineffective in cells expressing wild-type p53 activity. Therefore, the endogenous p53 status seems to be a major determinant for the effectiveness of a p53-based gene therapy for thyroid cancer.

Antibiotics, Antineoplastic↗
PubMed1999

Bivariate flow cytometry DNA/BrdUrd analysis of plant cell cycle.

We describe a protocol for flow cytometry analysis of cell cycle in plants using indirect immunolabelling staining and Vicia faba, Pisum sativum and Zea mays root tip cells as model systems. The protocol is based on simultaneous analysis of two fluorescent signals. The first, obtained after staining with propidium iodide, is used to quantify nuclear DNA content. The second, obtained after indirect immunofluorescent staining of bromodeoxyuridine (BrdUrd), is used to quantify the amount of BrdUrd incorporated into nuclear DNA. In an attempt to standardize the procedure, the effects of various conditions for partial DNA denaturation using HCl, as well as of BrdUrd concentration and incorporation time on flow cytometry DNA/BrdUrd content analysis have been studied. Maximum BrdUrd-linked fluorescence was observed after a 30 min pulse with 10 microM BrdUrd and after DNA denaturation with 1.5 N HCl (final concentration) for 30 min at 25 degrees C. Under these conditions, DNA content histograms with relatively small coefficient of variation (< 4%, full peak) could be obtained. To avoid non-specific staining of cytoplasm and cell walls, the protocol involves the use of nuclei isolated from formaldehyde-fixed tissues. Fixed isolated nuclei are stable and may be stored in hexylene glycol 0.75 M at 4 degrees C for prolonged periods prior to actual staining and analysis.

Bromodeoxyuridine↗
PubMed1999

Epitope mapping of the monoclonal antibody MM12.10 to external MDR1 P-glycoprotein domain by synthetic peptide scanning and phage display technologies.

Epitope mapping of MDR1-P-glycoprotein using specific monoclonal antibodies (mAbs) may help in delineating P-glycoprotein topology and hence in elucidating the relationship between its structural organization and drug-efflux pump function. In this work, by using synthetic peptide scanning and phage display technologies, the binding sites of the mAb MM12.10, a novel antibody to intact human multidrug resistant (MDR) cells, were studied. The results we obtained confirm that two regions localized on the predicted fourth and sixth loops are indeed external and that MDR1 peptides covering the inner domain of the current 12 transmembrane segment (TMs) model of P-glycoprotein could form part of the MM12.10 epitope.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Study on mutations and antiretroviral therapy (SMART): preliminary results.

Resistance to antiretroviral drugs is believed to be an important cause of treatment failure in human immunodeficiency virus (HIV)-infected patients, however, the role of susceptibility assays in the management of these individuals needs to be defined. SMART (study on mutations and antiretroviral therapy) is an ongoing study on mutations and antiretroviral therapy focused particularly on HIV-infected patients treated with two nucleoside analogue reverse transcriptase inhibitors (NRTIs). Plasma HIV-1 RNA was assessed by NASBA (nucleic acid sequence-based amplifications) (Organon Teknika, Boxtel, The Netherlands) with a detection limit of 80 copies/ml, whereas resistance was assessed by direct sequencing of the RT pol gene in patients with detectable viraemia, and by Antivirogram (Virco) in non-responder patients. The preliminary results of this study show that both genotypic and phenotypic assays identify mutated viral strains in the majority of patients failing a dual regimen. Furthermore, the data indicate a high rate of genotypic resistance to lamivudine in both responders and non-responders, a high rate of phenotypic resistance to lamivudine in non-responders, no genotypic resistance to didanosine and stavudine in responders, and a very low rate of both genotypic and phenotypic resistance to didanosine and stavudine in non-responders.

Anti-HIV Agents↗
PubMed1998

Nuclear factor kB-independent cytoprotective pathways originating at tumor necrosis factor receptor-associated factor 2.

Most normal and neoplastic cell types are resistant to tumor necrosis factor (TNF) cytotoxicity unless cotreated with protein or RNA synthesis inhibitors, such as cycloheximide and actinomycin D. Cellular resistance to TNF requires TNF receptor-associated factor 2 (TRAF2), which has been hypothesized to act mainly by mediating activation of the transcription factors nuclear factor kB (NFkB) and activator protein 1 (AP1). NFkB was proposed to switch on transcription of yet unidentified anti-apoptotic genes. To test the possible existence of NFkB-independent cytoprotective pathways, we systematically compared selective trans-dominant inhibitors of the NFkB pathway with inhibitors of TRAF2 signaling for their effect on TNF cytotoxicity. Blockade of TRAF2 function(s) by signaling-deficient oligomerization partners or by molecules affecting TRAF2 recruitment to the TNF receptor 1 complex completely abrogated the cytoprotective response. Conversely, sensitization to TNF cytotoxicity induced by a selective NFkB blockade affected only a fraction of TNF-treated cells in an apparently stochastic manner. No cytoprotective role for c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), which are activated by TRAF2 and contribute to stimulation of activator protein 1 activity, could be demonstrated in the cellular systems tested. Although required for cytoprotection, TRAF2 is not sufficient to protect cells from TNF + cycloheximide cytotoxicity when overexpressed in transfected cells, thus indicating an essential role of additional TNF receptor 1 complex components in the cytoprotective response. Our results indicate that TNF-induced cytoprotection is a complex function requiring the integration of multiple signal transduction pathways.

Animals↗
PubMed1998

Apoptotic, non-apoptotic, and anti-apoptotic pathways of tumor necrosis factor signalling.

Early events in the signalling of tumor necrosis factor-receptor 1 (TNF-R1), which is the main TNF receptor on most cell types, have been clarified recently. A multimolecular signal transducing complex from which several pathways originate rapidly forms upon TNF-induced aggregation of the receptor. Although fully capable of transducing apoptotic signals, which depend on the adapter Fas-associated death domain protein (FADD) and on the subsequent recruitment/activation of the apoptotic proteases, TNF-R1 usually does not kill cells; this is due to the induction of a complex cytoprotective response that requires TNF-receptor associated factor 2 (TRAF2), a signal transducer that couples TNF-R1 to both nuclear factor kappaB (NFkappaB)-dependent and NFkappaB-independent transcriptional events implicated in induction of genes protecting from TNF cytotoxicity. Although absolutely required for cytoprotection, TNF-receptor associated factor 2 is not sufficient to protect cells from TNF, thus suggesting that it may act in concert with additional TNF-R1 complex components. In this commentary, we will discuss some critical aspects of TNF-R1 signal transduction that are not fully understood: Why do cells not die before the protective protein synthesis has occurred? What are the mechanisms implicated in the termination of each TNF-R1-elicited response? Are there regulatory mechanisms capable of influencing the composition of the TNF-R1 complex and, consequently, the propagation of specific signals?

Animals↗
PubMed1998

Combined impairment of nutritional parameters and thyroid homeostasis in mildly iodine-deficient children.

The relation between thyroid homeostasis and the biochemical parameters of subclinical protein malnutrition has been analyzed in schoolchildren in a rural area in the south of Italy, known to be moderately iodine-deficient. The sera of 32 children (15 males and 17 females aged 6 to 11 years) have been analyzed. These children were divided into two groups, according to thyroid function: (1) 16 euthyroid children (mean thyrotropin [TSH] 2.38 +/- .35 mU/L; 6 with goiter) and (2) 16 subclinical hypothyroid children (mean TSH 7.32 +/- 1.68 mU/L; 6 with goiter). Retinol circulating complex (RCC) components were determined in serum by high-performance liquid chromatography (HPLC) and radial immunodiffusion and the essential and nonessential amino acid levels by ion exchange chromatography. Reduced retinol binding protein (RBP) and transthyretin (TTR) levels were recorded in the sera of 11 of 32 (34%) and in 5 of 32 (16%) patients, respectively. The linear regression analysis revealed that RBP and TSH levels were inversely correlated (r = -0.514; p < 0.0026). The RBP levels were subnormal in 2 of 16 euthyroid and in 9 of 16 hypothyroid patients (Fisher test p < 0.023), and the mean RBP levels were significantly reduced in the hypothyroid patients when compared with those of the euthyroid group (p < 0.0026). The retinol/RBP ratio was also significantly different between euthyroid and hypothyroid children (0.75 vs. 0.95; p < 0.0002). The mean essential amino acid levels, with the exception of methionine, were all in the normal range. The selected amino acid ratios confirmed that the patients were exposed to mild protein malnutrition. These results provide evidence that even mild protein-energy malnutrition may have detrimental effects on thyroid homeostasis in iodine-deficient areas.

Amino Acids↗
PubMed1998

Orphan receptor hepatocyte nuclear factor-4 antagonizes estrogen receptor alpha-mediated induction of human coagulation factor XII gene.

Factor XII (FXII) is a liver-specific zymogen involved in the regulation of hemostasis, particularly in the activation of fibrinolysis. Transcription of the FXII gene is stimulated by estrogens through specific interaction of the estrogen receptor alpha (ER alpha) with an estrogen response element present on FXII promoter. Interestingly, the magnitude of ER alpha induction in liver HepG2 cells is much lower than in NIH3T3 fibroblasts, suggesting that cell-specific factors may modulate ER alpha-dependent trans-activation. Comparative footprinting analysis of FXII promoter (from nucleotides -181 to +49) in liver vs. non-liver cell environments allowed identification of four deoxyribonuclease I-protected sites only in the presence of HepG2 nuclear extracts. Computerized homology search identified sites III and IV as consensus binding sequences for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4), formerly an orphan receptor belonging to the superfamily of steroid/thyroid hormone nuclear receptors. In transient transfection assays in NIH3T3 cells, HNF-4 significantly inhibited (70%) estrogen induction of FXII promoter while not affecting basal promoter activity. Conversely, HNF-4 did not inhibit estrogen inducibility of FXII promoter in HepG2 cells due to the high endogenous levels of HNF-4 protein. In gel shift assays, HNF-4, either present in HepG2 nuclear extracts or generated by in vitro transcription/translation, specifically bound FXII promoter. This interaction is strictly required in eliciting the antagonistic effect because in NIH3T3 cells, selective mutations of sites III and IV abrogated HNF-4 inhibitory properties. In the liver-specific environment, the same mutant construct exhibited higher estrogen-dependent inducibility compared with native promoter. Rescue of estrogen responsiveness was also achieved using a dominant negative HNF-4, which counteracted endogenous HNF-4 activity. In conclusion, our findings address a direct role for HNF-4 in modulating estrogen-dependent transcription of the FXII gene promoter.

Animals↗
PubMed1997

Tumor necrosis factor (TNF) receptor 1 signaling downstream of TNF receptor-associated factor 2. Nuclear factor kappaB (NFkappaB)-inducing kinase requirement for activation of activating protein 1 and NFkappaB but not of c-Jun N-terminal kinase/stress-activated protein kinase.

Like other members of the tumor necrosis factor (TNF) receptor family, p55 TNF receptor 1 (TNF-R1) lacks intrinsic signaling capacity and transduces signals by recruiting associating molecules. The TNF-R1 associated death domain protein interacts with the p55 TNF-R1 cytoplasmic domain and recruits the Fas-associated death domain protein (which directly activates the apoptotic proteases), the protein kinase receptor interacting protein, and TNF receptor-associated factor 2 (TRAF2). TRAF2 has previously been demonstrated to activate both transcription factor nuclear factor kappaB (NFkappaB) and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, which in turn stimulates transcription factor activating protein 1 (AP1) mainly via phosphorylation of the c-Jun component. We have investigated the signaling properties of NFkappaB-inducing kinase (NIK), a TRAF2-associated protein kinase that mediates NFkappaB induction. NIK was found to be unable to activate JNK/SAPK, mitogen-activated protein kinase, or p38 kinase. Moreover, NIK was not required for JNK/SAPK activation by TNF-R1, thus representing the first TNF-R1 complex component to dissect the NFkappaB and the JNK/SAPK pathways. Despite being unable to activate JNK/SAPK and mitogen-activated protein kinase, NIK strongly activated AP1 and was required for TNF-R1-induced AP1 activation. Therefore, NIK links TNF-R1 to a novel, JNK/SAPK-independent, AP1 activation pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

p53 re-expression inhibits proliferation and restores differentiation of human thyroid anaplastic carcinoma cells.

Alterations of the tumor suppressor gene p53 are uncommon in differentiated thyroid neoplasia but are detected at high frequency in anaplastic thyroid carcinoma suggesting that impaired p53 function may contribute to the undifferentiated and highly aggressive phenotype of these tumors. Effects of wild type p53 (wt-p53) re-expression were investigated in a human anaplastic thyroid carcinoma cell line (ARO) expressing a mutated p53. ARO cells were stably transfected with the temperature-sensitive p53 Val135 gene (ts-p53) which exhibits wild type-like activity at 32 degrees C. Exogenous wt-p53 function in ARO-tsp53 clones was assessed by evaluating its transcriptional activity on a CAT reporter vector containing p53 binding sites. At 32 degrees C, a significant reduction in the proliferation rate (approximately or equal to 50%) was observed, with accumulation of cells in the G0/G1 phase of the cell cycle. This effect was accompanied by induction of the expression of the growth inhibitor p21/Waf1 gene. At 32 degrees C, ARO-tsp53 clones also showed a marked impairment of their tumorigenic potential. Furthermore, transfected clones re-acquired the ability to respond to thyrotropin (TSH) stimulation showing an increased expression of thyroid-specific genes (thyroglobulin, thyroperoxidase and TSH receptor). In conclusion, re-expression of wt-p53 activity in ARO cells, inhibits cell proliferation and restores responsiveness to physiological stimuli.

Carcinoma↗
PubMed1991

Rapid and sensitive high-performance liquid chromatographic method for the analysis of tryptophan, tyrosine and phenylalanine in biological samples.

In order to overcome problems related to the determination of free tryptophan in biological fluids using conventional methods, we have developed an accurate and reliable procedure based on a specific pretreatment of samples followed by a very rapid and sensitive reversed-phase high-performance liquid chromatographic analysis. The pretreatment consists of adding to the samples of a very low amount of 3 M phosphate buffer to maintain pH in the physiological range followed by ultrafiltration. The precision, reproducibility and sensitivity of our method were also evaluated. The recovery of each amino acid was greater than 92%. The use of a microbore column allows the detection of up to 0.2 pmol/microliter of amino acid. The method has been applied to the analysis of samples obtained from 25 normal and 10 phenylketonuric subjects.

Chromatography, High Pressure Liquid↗
PubMed1990

Simultaneous high-performance liquid chromatographic determination of amino acids in a dried blood spot as a neonatal screening test.

A new screening test on dried blood spots for inherited disorders of amino acid metabolism using reversed-phase high-performance liquid chromatography (RP-HPLC) is described. The method allows the simultaneous analysis of fourteen different amino acids; among these, seven whose blood levels are increased in the most important amino acid disorders have been determined. The procedure requires a preliminary extraction of the amino acids from 9-mm autoclaved dried blood spots by sonication in phosphate-buffered saline. A precolumn o-phthaldialdehyde-3-mercaptopropionic acid derivatization is then followed by analysis of the amino acids by RP-HPLC. Blood-spots levels of histidine (His), tyrosine (Tyr), valine (Val), methionine (Met), isoleucine (Ile), phenylalanine (Phe) and leucine (Leu) can be determined in a single 15-min run, including column washing and regeneration. The minimum detectable amount of each amino acid is 0.5 pmol with a linear dose-response range between 1 and 10 microM. The recovery for all amino acids is greater than 70% except for Met (66%). Up to 20,000 samples/year can be processed on a single automated analytical line resulting in an estimated cost of about US$ 0.25/sample. The multiple diagnostic capacity, the low cost and the possibility of complete automation of the method make it suitable for primary perinatal screening of amino acid disorders.

Amino Acid Metabolism, Inborn Errors↗
PubMed1990

Hypertrophic obstructive cardiomyopathy and infective endocarditis: a report of seven cases and a review of the literature.

Seven cases of infective endocarditis (IE) in patients with hypertrophic obstructive cardiomyopathy (HOCM) are presented in this report. The previous literature is critically reviewed, and the following points are discussed: (a) IE complicates HOCM in 5-9% of cases; (b) anatomical and haemodynamic alterations of HOCM cause microtraumas on heart valves and the endocardium; the resulting endocardial lesions represent sites for bacterial seeding as well as other congenital or acquired heart disease; (c) prognosis is worse in patients with IE associated with HOCM than in patients with IE alone or associated with congenital heart disease; (d) the most frequently isolated organisms are saprophytes; (e) most patients were exposed to bacteraemias before the onset of IE.

Adolescent↗
PubMed1988

Rapid and sensitive method for high-performance liquid chromatographic analysis of pterins in biological fluids.

A rapid and sensitive high-performance liquid chromatographic (HPLC) method for the analysis of the most important urinary pterins is described. The method involves a preliminary sample oxidation to stabilize and convert pterins into their fluorescent forms and a purification by anion-exchange chromatography, followed by a short reversed-phase HPLC separation with fluorometric detection and quantitation of the different pterins. A complete HPLC analysis is accomplished in as little as 15 min. The sensitivity of the method allows the detection of as little as 20 pg of each pterin with a mean recovery greater than 99% for all pterins analysed. Reference values were obtained from 50 normal babies aged between 1 and 120 days. A significant correlation was found between urinary biopterin levels and the age of the babies (r = 0.445), while neopterin did not show any significant correlation with age. The "biopterin neopterin creatinine ratio" (BNCR index) was also significantly correlated with the age of the babies (r = 0.428). This rapid and sensitive method for pterin determination in biological fluids may be useful in the differential diagnosis of the various hyperphenylalaninemic conditions identified by neonatal mass screening programmes.

Chromatography, High Pressure Liquid↗