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Biomedical subjects

G Federici

Publications and source records attributed to G Federici.

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

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.

Amino Acids↗
PubMed2000

Modulation of GST P1-1 activity by polymerization during apoptosis.

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.

Apoptosis↗
PubMed2000

Tissue factor and homocysteine levels in ischemic heart disease are associated with angiographically documented clinical recurrences after coronary angioplasty.

BACKGROUND: In ischemic heart disease (IHD) patients high plasma levels of Tissue Factor (TF), the trigger of coagulation cascade, are present. Homocysteine (Hcy) is a risk factor for coronary artery disease, and several different pathophysiological mechanisms by which Hcy may play a role in thrombus formation have been postulated in "in vitro" studies. We investigated the "in vivo" role of Hcy in affecting plasma levels of TF, its inhibitor Tissue Factor Pathway Inhibitor (TFPI) and hypercoagulability. METHODS AND RESULTS: We investigated 119 IHD patients who underwent PTCA and compared them with 103 healthy subjects. TF, TFPI, Thrombin-Antithrombin complexes (TAT) and Hcy levels were significantly higher in the patients than in the controls. A positive correlation was found between Hcy and TF (r = 0.54; p < 0.0001), Hcy and TFPI (r = 0.26; p <0.05) as well as Hcy and TAT (r = 0.33; p <0.0001) levels. An inverse correlation existed between folate intake and Hcy levels (r = -0.28; p = 0.001). Hcy levels within the first quartile and in the highest quartile were associated with a lower (p < 0.001) and higher (p <0.0001) rate of clinical recurrences, respectively. Patients with TF values in the first quartile had a lower rate of angiographically documented clinical recurrences as compared to those in the fourth quartile (p <0.01); those in the highest quartile of TF showed a higher rate of recurrences (p = 0.001). Multivariate analysis confirmed these results (first quartile of Hcy: OR 0.02, C1 0.002-0.27; fourth quartile of Hcy: OR 36.5, C1 3.6-365/first quartile of TF: OR 0.006, C1 0.001-0.44; fourth quartile of TF: OR 16.4, C1 3.0 - 90.0), also after adjustment for risk factors and Hcy and TF respectively. CONCLUSIONS: In this study we show that TF, TFPI and TAT levels are correlated with Hcy plasma levels in IHD patients, providing evidence of an "in vivo" pathophysiological mechanism of hyperhomocysteinemia. The observed association between angiographically documented clinical recurrences and TF and Hcy values awaits confirmation in studies designated to evaluate this issue on a larger number of patients.

Adult↗
PubMed1999

Modulation of glutathione transferase P1-1 activity by retinoic acid in neuroblastoma cells.

The ability of retinoic acid to modulate glutathione S-transferase P1-1 (GSTP1-1) activity has important implications both for cancer prevention and for anticancer therapy. We investigated GSTP1-1 expression and activity in the human neuroblastoma cell line SK-N-BE(2) (genotype A*/B*) under basal conditions and during 48-h incubation with 0.1 microM all-trans-retinoic acid. The steady-state levels of glutathione transferase P1-1 mRNA and protein during 48-h incubation with all-trans-retinoic acid did not increase substantially, but we detected a significant reduction of GSTP1-1 specific activity. This reduction in enzymatic activity could not be ascribed to a differential action of retinoic acid on the gene variants A* and B*; indeed, the two GSTP1-1 isoforms have different affinities toward 1-chloro-2,4-dinitrobenzene (CDNB), while we found a substantial invariance of the K(m) (CDNB) in the cytosol during retinoid treatment. A modulatory effect of retinoic acid on other enzymes involved in glutathione transferase P1-1 metabolism, such as the retinoic acid-induced tissue trans-glutaminase, might be hypothesized, as well as a direct inactivation of GSTP1-1 by the oxidative stress that characterizes the early phases of apoptosis.

Antineoplastic Agents↗
PubMed1999

Proton release on binding of glutathione to alpha, Mu and Delta class glutathione transferases.

Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k(1)>/=(2-5)x10(6) M(-1).s(-1)], which is slowly converted into the final Michaelis complex (k(2)=1100-1200 s(-1)). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.

Animals↗
PubMed1999

Extraction and determination of oxybutynin in human bladder samples by reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method is described for the determination of oxybutynin (OXB) in human bladder samples. Following homogenization, tissue samples underwent double extraction with hexane and eventually were concentrated by freeze-drying before analysis. Chromatographic separation was performed with a mobile phase of acetonitrile-water-1 M ammonium acetate, pH 7.0 (85:13:2, v/v/v) at a flow-rate of 0.5 ml/min and double (electrochemical and UV) detection was applied. The retention time of oxybutynin eluting peak was around 18 min. Using a standard curve range of 10 to 500 ng/ml the quantification limit with electrochemical detection was 5 ng/ml with an injection volume of 100 microl. Within-day and day-to-day relative standard deviation values were 4.9 and 9.81%, respectively, while a 94% accuracy and a 72% recovery was attained. We applied this method to compare the OXB levels into bladder wall tissue samples after passive diffusion and after electromotive drug administration (EMDA), using a two-chambered poly(vinyl chloride) diffusion cell designed and developed in our laboratory. The results obtained show that EMDA enhanced OXB penetration into bladder wall and that this novel way of local drug administration can be potentially used in patients with neurogenic bladder dysfunction or urinary incontinence.

Acetates↗
PubMed1999

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1-1 (GST P1-1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35 degrees C, glutathione (GSH) binding to GST P1-1 displays positive cooperativity, whereas negative cooperativity occurs below 25 degrees C. This binding mechanism minimizes changes of GSH affinity for GST P1-1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1-1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.

Adaptation, Physiological↗
PubMed1999

A heterozygous splice site mutation in COL6A1 leading to an in-frame deletion of the alpha1(VI) collagen chain in an italian family affected by bethlem myopathy.

Bethlem myopathy is a mild neuromuscular disorder with proximal muscular weakness and early flexion contractures. It is an autosomal dominant disease due to mutations in type VI collagen genes. We found a T-->C substitution at the +2 position of COL6A1 intron 14 in a family, leading to skipping of exon 14 and an in-frame deletion of 18 amino acids in the triple-helical domain of the alpha1(VI) collagen chain. The deletion included a cysteine residue believed to be involved in the assembly of type VI collagen dimers intracellularly, prior to the protein secretion. Analysis of the affected fibroblasts showed that the shortened alpha1(VI) collagen chains were synthesized but not secreted by the cells and that the amount of type VI collagen microfibrils deposited by the cells was reduced. The results suggest that the clinical phenotype is due to a reduction in the level of type VI collagen in the extracellular matrix.

Adult↗
PubMed1999

Cyclosporine A monitoring in children: abbreviated area under curve formulas and C2 level.

Cyclosporine A (CsA) is the most commonly used drug in organ transplantation. To evaluate exposure the gold standard is area under curve (AUC), but this is expensive and requires many blood samples. For this reason, abbreviated formulae have been developed in adults. Data in children are scanty. Formulae cannot be applied in patients taking CsA every 8 h. We tested the abbreviated AUC formula published for adults in renal transplant children on twice daily therapy and we propose a method, requiring three blood samples, for those taking CsA every 8 h. A very good correlation was obtained for both groups. Furthermore, we studied the correlation between CsA concentration 2 h after administration and AUC. A good correlation was found supporting the possible use of this parameter for monitoring CsA therapy.

Adolescent↗
PubMed1999

A novel de novo mutation in the triple helix of the COL6A3 gene in a two-generation Italian family affected by Bethlem myopathy. A diagnostic approach in the mutations' screening of type VI collagen.

Bethlem myopathy is an autosomal dominant inherited disease producing a mild neuromuscular disorder, characterized mainly by muscular weakness and multiple joint contractures. Bethlem myopathy is caused by mutations in one of the three chains of collagen type VI. Here we report the clinical description and the molecular characterization of the defect in a two-generation Italian family in which a Gly-->Arg substitution disrupts the triple helix structure of the alpha 3 chain of collagen type VI, an ubiquitous glycoprotein of the extracellular matrix. In this family the identification of the mutation also allowed one to exclude the disease in the grandfather. It is noteworthy that the father of the proband carries a de novo mutation, the first described for Bethlem myopathy.

Adult↗
PubMed1999

Serum homocysteine, MTHFR gene polymorphism, and carotid intimal-medial thickness in NIDDM subjects.

We assessed the contribution of serum homocysteine levels, an independent risk factor for vascular disease, and of the methylenetetrahydrofolate reductase (MTHFR) C677T mutation to the variability of carotid intimal-medial thickness (IMT) in patients with non-insulin-dependent diabetes mellitus (NIDDM). Ninety-five patients (33 males and 62 females, mean age 53 +/- 10 years) without nephropathy or other vascular complications were enrolled. Fasting total serum homocysteine and other biochemical analytes were measured. The MTHFR polymorphism was determined by the polymerase chain reaction. Common carotid IMT and plaques or stenoses in the carotid district were measured by ultrasonography. Serum total homocysteine concentrations were higher in subjects with the mutant (Val/Val) genotype than in those with the Ala/Val plus Ala/Ala genotypes (P = 0.02). On univariate analysis, carotid IMT was significantly associated with age, body mass index (BMI), systolic blood pressure, and total cholesterolemia. No significant association was found between IMT and serum homocysteine or the MTHFR polymorphism, although a slightly greater IMT was observed in the homozygous Val genotypes. On multiple regression analysis, only age and BMI were independently associated with IMT and explained about 40% of IMT variability. The results did not change when the analysis was restricted to the subgroups with or without atherosclerotic plaques in the carotid district. In 95 Italian NIDDM patients without nephropathy, neither basal levels of serum total homocysteine nor the MTHFR C677T polymorphism predicted significant changes in common carotid intimal-medial thickness.

Arteriosclerosis↗
PubMed1999

Influence of the human paraoxonase polymorphism (PON1 192) on the carotid-wall thickening in a healthy population.

BACKGROUND: Serum paraoxonase (PON1) is a high-density lipoprotein-bound enzyme that can prevent oxidation of low-density lipoprotein and thus exert an anti-atherogenic effect. A polymorphism at codon 192 (Gln/Arg) of the PON1 gene gives rise to two isoforms that differ in substrate-dependent activity. OBJECTIVE: To determine any independent contribution of this polymorphism to the variability of intimal-medial thickness (IMT) of the common carotid artery for a sample of asymptomatic adult subjects from southern Italy by ultrasonography. METHODS: We studied 196 unrelated asymptomatic subjects (mean age 55.1 years), drawn from participants in a cardiovascular-disease-prevention campaign. Plasma levels of lipids and glucose were measured by routine methods. PON1 polymorphism was determined by polymerase chain reaction. IMT was measured from high-resolution B-mode echo-Doppler ultrasonography images. RESULTS: Prevalences of alleles A (Gln) and B (Arg) were 0.68 and 0.32, respectively. We found no significant difference with regard to plasma levels of lipids and glucose and other variables among the PON1 genotypes, although subjects with BB had higher levels of triglycerides and lower levels of high-density lipoprotein cholesterol. Common carotid artery IMT was slightly greater in subjects with BB, although no significant association between PON1 genotypes and common carotid artery IMT was found, even after adjustment for confounding variables. CONCLUSIONS: Our findings demonstrate that there is no significant association between PON1 gene polymorphism at codon 192 and common carotid artery IMT for an Italian population. However, the fact that we found slightly greater IMT in subjects with genotype BB would suggest that the study should be performed again with a larger sample.

Adult↗
PubMed1998

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione.

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.

Alanine↗
PubMed1998

Shifting substrate specificity of human glutathione transferase (from class Pi to class alpha) by a single point mutation.

Substrate selectivity, among glutathione transferase (GST) isoenzymes, appears to be determined by a few residues. As part of study to determine which residues are class-specific determinants, Tyr 108 (an important residue of the class Pi) has been changed to a valine, the structural equivalent of a class Alpha enzyme. Using a panel of selected substrates, "diagnostic" for either class Pi or Alpha, it is shown here that this single mutation significantly alters the catalytic properties of the class Pi enzyme and shifts the substrate specificity of the enzyme toward that of the class Alpha enzyme.

Amino Acid Sequence↗
PubMed1998

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.

Binding Sites↗
PubMed1998

Evidence for an induced-fit mechanism operating in pi class glutathione transferases.

Three-dimensional structures of the apo form of human pi class glutathione transferase have been determined by X-ray crystallography. The structures suggest the enzyme recognizes its substrate, glutathione, by an induced-fit mechanism. Compared to complexed forms of the enzyme, the environment around the catalytic residue, Tyr 7, remains unchanged in the apoenzyme. This observation supports the view that Tyr 7 does not act as a general base in the reaction mechanism. The observed cooperativity of the dimeric enzyme may be due to the movements of a helix that forms one wall of the active site and, in particular, to movements of a tyrosine residue that is located in the subunit interface.

Apoenzymes↗