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Biomedical subjects

P Paoli

Publications and source records attributed to P Paoli.

At least 19 recordsLinked to original sources
PubMed2004

Insulin inhibits platelet-derived growth factor-induced cell proliferation.

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

Animals↗
PubMed2001

Addition of small molecules by Zn(II) and Cu(II) dinuclear complexes obtained by an amino-phenolic ligand. Crystal structures of the dinuclear zinc complex assembling butanolate and azide anions.

The coordination properties of the ligand 2,6-bis-[[bis-(2-aminoethyl)-amino]methyl]-phenol (L) toward the zinc ion were determined by means of potentiometric measurements in aqueous solution (298.1 +/- 0.1 K, I = 0.15 mol dm(-3)). L forms mono- and dinuclear complexes with zinc; the stable dinuclear complexes are practically the only existing species using L/Zn(II), molar ratio of 1:2. In these species, each dien subunit binds one zinc ion, while the phenolate moiety bound to both ions allows the two metal centers to be in close proximity with an incomplete coordination environment. The role of this metal-metal distance in binding secondary ligands was investigated for the dinuclear systems 2Zn/L and 2Cu/L by potentiometric (298.1 +/- 0.1 K, I = 0.15 mol dm(-3)) and NMR experiments in aqueous and methanol solution with a series of small guests having nitrogen donor atoms. The coordination sphere of the two metals was completed by adding 1 equiv of only those guests, which showed at least two contiguous donor atoms or two lone pairs on the same atom, to exactly fit the metal-metal distance without modifying the metal cluster. To do this, the imidazole molecule which shows the highest addition constants to the [M(2)H(-1)L](3+) species probably forms a mu-1,1-amino. These results are in agreement with the two crystal structures reported herein [Zn(2)(H(-1)L)(CH(3)CH(2)CH(2)CH(2)O)](ClO(4))(2) (space group P2(1)nb, a = 11.483(5), b = 14.166(5), c = 18.279(5) A, V = 2973(19) A(3), Z = 4, R = = 0.0701, wR2 = 0.1611) and [Zn(2)(H(-1)L)N(3)](ClO(4))(2) (space group C 2/c, a = 14.460(3), b = 12.814(3), c = 14.875(3) A, beta = 105.35(2) degrees, V = 2658(10) A(3), Z = 4, R = 0.0783, wR2 = 0.1806). In fact, these structures display a butanolate or azide guest linked to both Zn(II) ions of the dinuclear complex, resulting in a mu-1,1-oxo and mu-1,1-azido bridge, respectively.

Journal Article↗
PubMed2001

Hydrogen peroxide triggers the formation of a disulfide dimer of muscle acylphosphatase and modifies some functional properties of the enzyme.

Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.

Acid Anhydride Hydrolases↗
PubMed2000

Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities.

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the gamma-phosphate group of nucleoside triphosphates, and then attacks the beta-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In contrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated k(cat) values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg(2+) ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 degrees C, in the presence of millimolar Mg(2+) concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.

Acid Anhydride Hydrolases↗
PubMed2000

Polyamine macrocycles incorporating a phenolic function: their synthesis, basicity, and coordination behavior toward metal cations. Crystal structure of a binuclear nickel complex.

The synthesis and characterization of two new polyazamacrocycles, 1,4,7,10-tetraaza[12](2,6)phenolphane (L1) and 1,4,7,10,13-pentaaza[15](2,6)phenolphane (L2), are reported. Both ligands incorporate the 2,6-phenolic unit within the cyclic framework. The basicity behavior and the ligational properties of L1 and L2 toward Ni(II), Zn(II), and Cu(II) were determined by means of potentiometric measurements in aqueous solution (298.1 +/- 0.1 K, I = 0.15 mol dm-3). UV spectra were used to understand the role of the phenolic function in the stabilization of the cations. L1 and L2 behave as pentaprotic bases under the experimental conditions used. The UV spectra showed that the deprotonation of the phenolic function occurs at low pH values for both ligands, giving rise to the simultaneous presence of positive and (one) negative charges on the macrocycle. While L1 forms only mononuclear complexes, L2 can also form binuclear species with all the metal ions investigated. In the mononuclear species of both ligands, one nitrogen atom close to the phenol remains unbound. The UV spectra revealed that the phenol, bridging the two metal ions in phenolate form, plays an important role in the stabilization of the binuclear complexes of L2. The coordination sphere of the two metals is completed by adding a secondary ligand such as water molecules or OH-, in any case preferring substrates able to bridge the two close metal ions. These results are confirmed by the crystal structure of [Ni2(C16H28ON5)(H2O)2Cl2]Cl.H2O.CH3OH (space group P21/a, a = 14.821(5) A, b = 10.270(4) A, c = 17.663(6) A, beta = 108.87(3) degrees, V = 2544(2) A3, Z = 4, R1 = 0.0973, wR2 = 0.2136). This structure displays a Ni(II) binuclear complex of L2 in which the phenolic oxygen and a chlorine ion bridge the two close Ni(II) ions.

Journal Article↗
PubMed2000

Acylphosphatase is a strong apoptosis inducer in HeLa cell line.

Acylphosphatase (AcP) is a low-molecular-weight protein widely distributed in many vertebrate tissues with a yet unknown physiologic function. To study the in vivo behavior of AcP, HeLa cells were transiently transfected with a vector expressing the AcP/EGFP fusion protein. Analysis of the transfected cells showed a high level of cellular death in cells expressing the AcP/EGFP fusion protein with respect to control cells expressing EGFP alone. Flow cytometry and time lapse analysis of AcP/EGFP transfected cells evidenced a typical pattern of apoptosis. Surprisingly, cells transfected with a mutated form of AcP, with negligible in vitro acylphosphatase activity, undergo apoptosis as well as cells transfected with wild-type protein, suggesting that the physiologic role of AcP could be not related to this catalytic activity.

3T3 Cells↗
PubMed1999

The contribution of acidic residues to the conformational stability of common-type acylphosphatase.

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.

Acid Anhydride Hydrolases↗
PubMed1999

Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme.

The thermodynamics and kinetics of folding of common-type acylphosphatase have been studied under a variety of experimental conditions and compared with those of the homologous muscle acylphosphatase. Intrinsic fluorescence and circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-state mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Nevertheless, common-type acylphosphatase folds more rapidly, suggesting that the conformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate of common-type acylphosphatase is much higher than that of the muscle enzyme, indicating that the differences in conformational stability between the two proteins are primarily determined by differences in the rate of unfolding. The equilibrium m value is markedly different for the two proteins in the pH range of maximum conformational stability (5. 0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes similar to that of the muscle enzyme. Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and mu values, respectively) are notably reduced with respect to pH 5.5. The pH-induced decrease of the m value can be attributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme. This would decrease the positive net charge of the protein, leading to a greater compactness of the denatured state. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this change in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.

Acid Anhydride Hydrolases↗
PubMed1998

Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW-PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW-PTPs (called m-IF1 and m-IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m-IF2P) that presents three substitutions compared to m-IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m-IF1 and m-IF2. It is likely that a gene duplication process followed by mutations has generated this new gene.

3T3 Cells↗
PubMed1997

Mechanism of acylphosphatase inactivation by Woodward's reagent K.

The organ common-type (CT) isoenzyme of acylphosphatase is inactivated by Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3'-sulphonate) at pH6.0. The inactivation reaction follows apparent pseudo first-order kinetics. The dependence of the reciprocal of the pseudo first-order kinetic constant (kobs) on the reciprocal WRK concentration reveals saturation kinetics, suggesting that the WRK forms a reversible complex with the enzyme before causing inactivation. Competitive inhibitors, such as inorganic phosphate and ATP, protect the enzyme from WRK inactivation, suggesting that this reagent acts at or near to the enzyme active site. The reagent-enzyme adduct, which elicits a strong absorption band with lambdamax at 346 nm, was separated from unreacted enzyme by reverse phase HPLC and the modified protein was cleaved with endoproteinase Glu-C to produce fragments. The HPLC fractionation gave two reagent-labelled peptides (peak 1 and peak 2) that were analysed by ion-spray MS and sequenced. The former is VFFRKHTQAE (residues 20-29 of human CT acylphosphatase) and the latter IFGKVQGVFFRKHTQAE (residues 13-29). MS demonstrated that both peptides are WRK adducts. A fragment ion with m/z of 1171, which is present in the mass spectrum of peak 1, has been identified as a WRK adduct of the peptide fragment 20-26. The lambdamax at 346 nm of WRK adduct suggests that the modified residue is His-25. Five recombinant enzymes mutated in residues included in the 20-29 polypeptide stretch have been produced. Analysis of their reactivities with WRK demonstrates that His-25 is the molecular target of the reagent as its modification causes the inactivation of the enzyme. Since both His-25-->Gln and His-25-->Phe mutants maintain high catalytic activity, we suggest that the observed enzyme inactivation is caused by the reagent (covalently bound to His-25), which shields the active site.

Acid Anhydride Hydrolases↗
PubMed1997

19-nor-10-azasteroids, a new class of steroid 5 alpha-reductase inhibitors. 2. X-ray structure, molecular modeling, conformational analysis of 19-nor-10-azasteroids and comparison with 4-azasteroids and 6-azasteroids.

19-Nor-10-azasteroids are a new class of 5 alpha-reductase inhibitors whose activity depends on the presence of the bridgehead N-10 atom conjugated with the 4-en-3-one moiety in the A ring. The X-ray structure of 19-nor-10-azasteroid 1 has been determined and it is compared with the X-ray structure of testosterone. A complete conformational analysis of these compounds has been performed, determining the number and energy of the possible conformers, as well as the molecular flexibility of the 10-azasteroidal skeleton. Thus, MM2* molecular mechanics calculations and AM1 semiempirical energy refinements revealed that 19-nor-10-azasteroids 1-3 have four possible conformations with very small energy differences and that they are very flexible molecules. The conformational analysis has been extended to testosterone (4), which also showed conformational flexibility, with three different conformations, and to 6-azasteroid 5 and 4-azasteroid 6, for which only two thermally accessible conformations have been found. Compared to 19-nor-10-azasteroids 1-3, azasteroids 5 and 6 appear to be more rigid structures. By a best fit analysis of all conformers of 1-5 with the global minimum of testosterone (4-I) it has been found that the lowest energy conformers of 1, 3, and 5 are very close to the structure of 4-I, and among the conformers of 2, the best similarity has been observed for the highest energy conformer 2-IV.

5-alpha Reductase Inhibitors↗
PubMed1997

Common-type acylphosphatase: steady-state kinetics and leaving-group dependence.

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 degrees C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O-inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. all these findings, as well as the strong dependence of kcat upon the leaving group pK1, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.

Acid Anhydride Hydrolases↗
PubMed1997

Dynamics of HIV-1 mRNA expression in patients with long-term nonprogressive HIV-1 infection.

A large number of evidences indicate that progression of HIV disease is driven by an increase in viral burden. It is still unclear, however, to what extent this is contributed by the dysregulation of the molecular mechanisms governing virus gene expression at the transcriptional or posttranscriptional levels. To address this issue, several quantitative virologic parameters (including provirus transcriptional activity and splicing pattern) were analyzed in individuals with nonprogressive HIV infection and compared with those of a matched group of progressor patients. Exact quantification was achieved by a competitive PCR procedure using a multicompetitor template. Nonprogressors were characterized by striking differences in the levels of viremia, provirus copy number, and overall levels of all viral mRNA classes in peripheral blood mononuclear cells. Additionally, the transcriptional activity of the proviral DNA in these patients was mainly engaged in the production of multiprocessed transcripts, with a pattern resembling the early phases of the experimental infection. Taken together, these results show that both viral load and provirus transcription pattern are remarkably different in infected individuals nonprogressing toward overt disease, and further support the notion that disease progression is accompanied by a change in the kinetics of HIV gene expression.

Adult↗
PubMed1995

2-Methoxybenzoyl phosphate: a new substrate for continuous fluorimetric and spectrophotometric acyl phosphatase assays.

A new aromatic acyl phosphate, 2-methoxybenzoyl phosphate, has been synthesized. The compound shows an intrinsic fluorescence; it displays an intense emission band at 390 nm upon excitation in the near UV region. This band practically disappears after hydrolysis of the product. On the other hand, the product displays differences in the near UV absorption spectra measured before and after hydrolysis. The delta epsilon at 301 nm is 2720 M-1 cm-1, a value that is 4.3-fold higher than that of benzoyl phosphate (the usual substrate for acylphosphatase assay) at 283 nm. The main kinetic parameters of three different acylphosphatase molecular forms (the muscular isoenzyme and two subtypes of the organ common isoenzyme) were determined using both benzoyl phosphate and 2-methoxybenzoyl phosphate as substrates, and then compared. These kinetic data and the UV absorption and fluorescence properties of 2-methoxybenzoyl phosphate suggest that this compound has better substrate features than benzoyl phosphate, and can be used for both high sensitivity continuous fluorimetric and UV absorption spectrophotometric assays of acylphosphatase.

Acid Anhydride Hydrolases↗
PubMed1995

Synthesis and analytical profile of the new potent antibronchospastic agent 7-[(2,2-dimethyl)propyl]-1-methyl xanthine.

7-[(2,2-Dimethyl)propyl]-1-methyl xanthine (CAS 155006-67-0, MX2/120) is a new potent antibronchospastic agent with negligible side effects. The synthesis involves the alkylation of 3-benzyl-1-methyl xanthine with neopentyl bromide followed by removal of the benzyl protecting group. Main physico-chemical properties were determined using NMR, MS, IR, UV spectra and X-ray crystal structure. A quantitative determination of substances related to MX2/120 and of residual solvents by means of HPLC and GC, respectively, is described. MX2/120 is safely stored at room temperature for a long time.

Bronchodilator Agents↗
PubMed1994

Absolute configuration and biological activity of mequitamium iodide enantiomers.

The enantiomers of 1-methyl-3-(10H-phenothiazine-10-ylmethyl)-1-azoniabicyclo[2 ,2,2]octane iodide (1) were prepared by chiral chromatographic resolution of the precursor mequitazine (2). The (+)-(S)-enantiomer 1b is 10-fold more potent than (-)-(R)-enantiomer 1a as a histamine antagonist, while the two enantiomers show the same antimuscarinic activity in vitro. The absolute configuration of the more active dextrorotatory isomer has been determined by X-ray analysis. Conformational analysis and molecular modeling suggest that the (+)-(S)-enantiomer can adopt a conformation similar to that attributed to the receptor binding conformers of classical antihistamines.

Acetylcholine↗
PubMed1994

Synthesis, characterization, and analytical studies of adosupine, a potential new drug for urinary incontinence.

The synthesis and the spectroscopic characterization of a new potential drug for urinary incontinence, adosupine, is described. Adosupine and its potential synthesis impurities were analyzed by a new HPLC method that was developed with a C18 reversed-phase column. The analysis was made under isocratic conditions, with a mobile phase of acetonitrile:water (15:85, v/v). Resolution of all synthesis impurities was allowed. The method was also applied to stability studies of adosupine in solid state and in solution under different conditions. With the conditions used, only one degradation product was shown by HPLC analysis; it was isolated, characterized, and identified as the hydrolysis product of the lactam ring present in the adosupine structure.

Chromatography, High Pressure Liquid↗