Biomedical subjects

A Desideri

Publications and source records attributed to A Desideri.

At least 55 records · Page 3Linked to original sources

PubMed2000

Single mutation at the intersubunit interface confers extra efficiency to Cu,Zn superoxide dismutase.

The Val28-->Gly single mutant at the subunit interface of Cu,Zn superoxide dismutase from Photobacterium leiognathi displays a k(cat)/K(M) value of 1.7x10(10) M(-1) s(-1), twice that of the native enzyme. Analysis of the three-dimensional structure indicates that the active site Cu,Zn center is not perturbed, slight structural deviations being only localized in proximity of the mutation site. The enzyme-substrate association rate, calculated by Brownian dynamics simulation, is identical for both enzymes, indicating that the higher catalytic efficiency of the Val28-->Gly mutant is not due to a more favorable electrostatic potential distribution. This result demonstrates the occurrence of an intramolecular communication between the mutation site and the catalytic center, about 18 A away and indicates a new strategy to encode extra efficiency within other members of this enzymatic family.

Amino Acid Substitution↗

PubMed2000

Prognostic usefulness of dobutamine-induced ST-segment elevation and T-wave normalization after uncomplicated acute myocardial infarction.

We followed 229 consecutive patients exhibiting negative T waves on infarct-related electrocardiographic leads; these patients underwent dobutamine stress echocardiography within 10 days after a first uncomplicated acute myocardial infarction. T-wave normalization, but not ST-segment elevation, recognized patients at higher risk of cardiac events and optimized the prognostic accuracy of both myocardial viability and ischemia, to which it was correlated and became an independent predictor in cases of subdiagnostic stress echocardiography.

Disease-Free Survival↗

PubMed2000

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene.

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

Amino Acid Sequence↗

PubMed2000

Noninvasive risk stratification in women with uncomplicated acute myocardial infarction.

The aim of our study was to compare the prognostic value of stress echocardiography and exercise electrocardiography after uncomplicated non-Q-wave acute myocardial infarction in a series of 89 female patients. Our data show that stress echocardiography has independent predictive value in a female patient population recovering from uncomplicated acute myocardial infarction.

Aged↗

PubMed2000

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system.

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E(o) = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information for a deeper understanding of cytochrome c folding and structural/functional organization.

Animals↗

PubMed2000

Single mutation induces a metal-dependent subunit association in dimeric Cu,Zn superoxide dismutase.

Tryptophan 83, a residue strongly involved in the intersubunit interaction of the Cu,Zn superoxide dismutases from Photobacterium leiognathi, has been selectively mutated to phenylalanine or tyrosine. The recombinant mutant enzymes expressed in Escherichia coli were purified in two well distinct and stable forms, one dimeric and fully active and the other monomeric and devoid of metals. In agreement, in vitro experiments indicate that the removal and addition of zinc in the mutant enzymes induces monomerization and dimerization, respectively, while does not perturb the dimeric association of the native protein. This is the first unambiguous experimental proof of a direct communication between the intersubunit interface and the metal active site.

Base Sequence↗

PubMed2000

Role of the tertiary and quaternary structures in the stability of dimeric copper, zinc superoxide dismutases.

The equilibrium unfolding process of human Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism and fluorescence spectroscopy as a function of increasing guanidinium hydrochloride concentration. The process occurs through the formation of a monomeric intermediate species following a three-state transition equilibrium. Comparison with the stability of the prokaryotic Cu,Zn SOD from P. leiognathi shows that the eukaryotic enzyme is more stable than the prokaryotic enzyme by approximately 3 kcal/mol. This difference is due to the monomer-to-unfolded equilibrium, while the dimer-to-monomer equilibrium is comparable for the two enzymes despite their different intersubunit interactions. These results are confirmed by the unfolding of the copper-depleted derivatives. The Cu,Zn superoxide dismutase represents a good example of how evolution has found two independent quaternary assemblies maintaining the same dimer stability.

Circular Dichroism↗

PubMed2000

Molecular dynamics simulation of solvated azurin: correlation between surface solvent accessibility and water residence times.

A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent-accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67.

Azurin↗

PubMed2000

Structure and hydration of BamHI DNA recognition site: a molecular dynamics investigation.

The results of a 3-ns molecular dynamics simulation of the dodecamer duplex d(TATGGATCCATA)(2) recognized by the BamHI endonuclease are presented here. The DNA has been simulated as a flexible molecule using an AMBER force field and the Ewald summation method, which eliminates the undesired effects of truncation and permits evaluation of the full effects of electrostatic forces. The starting B conformation evolves toward a configuration quite close to that observed through x-ray diffraction in its complex with BamHI. This configuration is fairly stable and the Watson-Crick hydrogen bonds are well maintained over the simulation trajectory. Hydration analysis indicates a preferential hydration for the phosphate rather than for the ester oxygens. Hydration shells in both the major and minor groove were observed. In both grooves the C-G pairs were found to be more hydrated than A-T pairs. The "spine of hydration" in the minor groove was clear. Water residence times are longer in the minor groove than in the major groove, although relatively short in both cases. No special long values are observed for sites where water molecules were observed by x-ray diffraction, indicating that water molecules having a high probability of being located in a specific site are also fast-exchanging.

Base Pairing↗

PubMed2000

Effects of left bundle branch block on myocardial FDG PET in patients without significant coronary artery stenoses.

UNLABELLED: Cardiac PET studies in patients with left bundle branch block (LBBB) are few, and the results are conflicting. In particular, even if a reduced uptake of FDG is reported, confirmation in a large group of patients and exact understanding of the underlying cause are lacking. METHODS: We selected 29 consecutive patients who had complete LBBB and no significant stenosis on coronary angiography scheduled for FDG and 13N-NH3 PET for myocardial viability evaluation at our center. Wall motion was evaluated using 2-dimensional echocardiography. Ten volunteers without coronary stenosis or LBBB served as a control group. RESULTS: All LBBB patients had a reverse mismatch in the septum, defined as reduced uptake of FDG in comparison with 13N-NH3. The mismatch extended to the anterior and inferior walls in 17 patients. The mean (+/-SD) septal-to-lateral ratio was 0.57 +/- 0.11 for FDG (range, 0.28-0.76) and 0.99 +/- 0.12 for 13N-NH3 (range, 0.75-1.18), with P < 0.0001. In contrast, no significant differences in uptake were seen in the control group, which had a septal-to-lateral ratio of 0.95 +/- 0.13 for FDG (range, 0.78-1.15; P < 0.01 with respect to LBBB patients) and 0.94 +/- 0.11 (range, 0.85-1.20) for 13N-NH3. CONCLUSION: Our study suggests that in LBBB patients without significant coronary stenosis, FDG uptake in the septum changes without a correlating change in perfusion. To avoid possible overestimation of necrosis, especially in the LAD territory, this phenomenon must be considered in evaluations of myocardial viability using FDG images.

Adult↗

PubMed2000

Evidence of walk-through phenomenon during echocardiographic dipyridamole stress test.

This case report deals with induced regional wall motion abnormalities that spontaneously disappeared during an echocardiographic stress test with dipyridamole. A patient underwent this test because of atypical chest discomfort and a positive result of exercise stress test. Transient septal, apical and anterior akinesia were observed after the first dose of dipyridamole, but they were short-lasting and did not return during the continuation of the test. Coronary angiography showed a critical stenosis of the left coronary artery. A mechanism similar to that responsible for the walk-through phenomenon might explain the observed findings. Thus stress echo with dipyridamole needs careful continuous monitoring, because transient wall motion abnormalities can otherwise be missed resulting in a false negative test.

Coronary Angiography↗

PubMed1999

Molecular dynamics simulations of Cu,Zn superoxide dismutase: effect of temperature on dimer asymmetry.

Molecular dynamics simulations of solvated dimeric Cu,Zn superoxide dismutase have been carried out at four temperatures, namely 200, 225, 250 and 300 K. Analysis of the backbone-to-backbone hydrogen bonds number indicates that the symmetry observed in the two subunits at 200 K is gradually lost by heating the system. The C(alpha) atoms displacement cross-correlation maps confirm that the asymmetric behaviour of the two subunits increases as a function of temperature. The dynamic cross-correlation of the subunits volumes indicates a fast correlation between the two subunits at 300 K, which is delayed upon lowering the simulation temperature. These results indicate that temperature plays an essential role in injecting such an asymmetry; the two subunits being asymmetric and in rapid communication at 300 K, and almost symmetric and in slow communication at lower temperatures.

Journal Article↗

PubMed1999

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.

Circular Dichroism↗

PubMed1999

EPR detection of protein-derived radicals in the reaction of H(2)O(2) with Fe bound in mitochondrial F(1)ATPase.

A severe inactivation is obtained upon the addition of H(2)O(2) to bovine heart F(1)ATPase samples containing Fe(III) in the nucleotide-independent site, and Fe(II) in the ATP-dependent site. EPR spectra at 4.9 K of these samples indicate that H(2)O(2) produces the complete oxidation of Fe(II) to Fe(III) and the concomitant appearance of two protein-derived radical species. The two signals (g = 2.036 and g = 2.007) display a different temperature dependence and saturation behavior. The relaxation properties of the radical at g = 2.036 suggest magnetic interaction with one of the two iron centers. Such events are not observed when H(2)O(2) is added either to native F(1)ATPase containing a high amount of Fe(II) and low amount of Fe(III) or to F(1)ATPase deprived of endogenous Fe and subsequently loaded with only Fe(III) in both sites. It is hypothesized that in F(1)ATPase samples containing both Fe(III) and Fe(II), intramolecular long-range electron transfer may occur from Fe(II) to a high oxidation state species of Fe formed in the nucleotide-independent site upon oxidation of Fe(III) by H(2)O(2).

Animals↗

PubMed1999

Stress echocardiography and exercise electrocardiography for risk stratification after non-Q-wave uncomplicated myocardial infarction.

The aim of our study was to compare the prognostic value of stress echocardiography and exercise electrocardiography after uncomplicated non-Q-wave acute myocardial infarction in a series of 68 consecutive patients. Our data show that stress echocardiography and exercise electrocardiography offer similar prognostic information after uncomplicated non-Q-wave AMI.

Adult↗

PubMed1999

A model for the incorporation of metal from the copper chaperone CCS into Cu,Zn superoxide dismutase.

BACKGROUND: Recent studies have identified the human copper chaperone CCS as the presumed factor responsible for copper incorporation into superoxide dismutase (SOD). A lack of knowledge of the chaperone's three-dimensional structure has prevented understanding of how the copper might be transferred. RESULTS: The three-dimensional structure of CCS was homology modelled using the periplasmic protein from the bacterial mercury-detoxification system and the structure of one subunit of the human SOD dimeric enzyme as templates. On the basis of the three-dimensional model, a mechanism for the transfer of copper from CCS to SOD is proposed that accounts for electrostatic acceptor recognition, copper storage and copper-transfer properties. CONCLUSIONS: The proposed model identifies a path for copper transfer based on the presence of different metal sites characterized by sulphur ligands. Such a model permits the development of strategies able to interfere with copper incorporation in SOD, providing a possible way to prevent or arrest degeneration in the fatal motor neuron disorder amyotrophic lateral sclerosis.

Amino Acid Sequence↗

PubMed1999

Characterization of the spectroscopic properties of the Cu,Co cluster in a prokaryotic superoxide dismutase.

A Cu,Co derivative of the Cu,ZnSOD from Photobacterium leiognathi, in which cobalt has been selectively substituted for zinc, has been prepared and spectroscopically investigated. The derivative shows three bands in the visible region at 530, 566, and 600 nm when copper is in the oxidized state. Reduction or depletion of the copper ion produce a shift of the band absorbing at 600 to 590 nm because of the detachment from copper of the imidazolate bridging the two metals when copper is in the oxidized state. Numerous isotropically shifted 1H NMR lines are observed when copper is oxidized, confirming the presence of the imidazolate bridge between the two metals. Comparison of the optical and the NMR spectra with those observed for the eukaryotic enzyme reveals the occurrence of slight but unambiguous differences diagnostic of a different degree of distortion of the metal cluster between the prokaryotic and eukaryotic enzymes.

Cobalt↗

PubMed1999

Toward the engineering of a super efficient enzyme.

The catalytic activity of a mutant of Photobacterium leiognathi Cu, Zn superoxide dismutase in which the Glu59 residue, conserved in most bacterial variants of the enzyme, has been replaced by glutamine was investigated by pulse radiolysis. At neutral pH the enzyme was found to have a kcat/KM of 1.0 +/- 0.1 x 10(10) M-1s-1 the highest value ever found for any superoxide dismutase. Brownian dynamics simulation suggests that such a high value is due to an enhanced substrate attraction by the modified electric field distribution. The mutant is also characterized by an active-site widely accessible for the solvent, since iodide is able to interact with the copper atom with an affinity constant twice as high as that found in the native enzyme. The large solvent accessible surface of the copper site together with a favorable distribution of the protein-generated electric field gives rise to the most efficient enzyme ever found with activity close to the diffusion limit.

Amino Acid Substitution↗