Biomedical subjects

Alessandro Desideri

Publications and source records attributed to Alessandro Desideri.

At least 19 recordsLinked to original sources

PubMed2006

The different cleavage DNA sequence specificity explains the camptothecin resistance of the human topoisomerase I Glu418Lys mutant.

Yeast cells expressing the Glu418Lys human topoisomerase I mutant display a camptothecin resistance that slowly decreases as a function of time. Molecular characterization of the single steps of the catalytic cycle of the purified mutant indicates that it has a relaxation activity identical to the wild-type protein but a different DNA sequence specificity for the cleavage sites when compared to the wild-type enzyme, as assayed on several substrates. In particular the mutant has a low specificity for CPT sensitive cleavable sites. In fact, the mutant has, at variance of the wild-type enzyme, a reduced preference for cleavage sites having a thymine base in position -1 of the scissile strand. This preference, together with the strict requirement for a thymine base in position -1 for an efficient camptothecin binding, explains the temporary camptothecin resistance of the yeast cell expressing the mutant and points out the importance of the DNA sequence in the binding of the camptothecin drug.

Amino Acid Substitution↗

PubMed2006

NMR structure of the p63 SAM domain and dynamical properties of G534V and T537P pathological mutants, identified in the AEC syndrome.

The p63 protein is crucial for epidermal development, and its mutations cause the extrodactyly ectodermal dysplasia and cleft lip/palate syndrome. The three-dimensional solution structure of the p63 sterile alpha-motif (SAM) domain (residues 505-579), a region crucial to explaining the human genetic disease ankyloblepharonectodermal dysplasia-clefting syndrome (AEC), has been determined by nuclear magnetic resonance spectroscopy. The structure indicates that the domain is a monomer with the characteristic five-helix bundle topology observed in other SAM domains. It includes five tightly packed helices with an extended hydrophobic core to form a globular and compact structure. The dynamics of the backbone and the global correlation time of the molecule have also been investigated and compared with the dynamical properties obtained through molecular dynamics simulation. Attempts to purify the pathological G534V and T537P mutants, originally identified in AEC, were not successful because of the occurrence of unspecific proteolytic degradation of the mutated SAM domains. Analysis of the structural dynamic properties of the G534V and T537P mutants through molecular dynamics simulation and comparison with the wild type permits detection of differences in the degree of freedom of individual residues and discussion of the possible causes for the pathology.

Abnormalities, Multiple↗

PubMed2005

New hints on the Ph-driven tautomeric equilibria of the topotecan anticancer drug in aqueous solutions from an integrated spectroscopic and quantum-mechanical approach.

The equilibria between the different forms of the topotecan anticancer drug have been studied at moderately acidic and physiological pH by an integrated computational tool rooted in the density functional theory and its time-dependent extension together with the polarizable continuum model. The results allow an unbiased selection between the different possible tautomeric forms and provide invaluable complements to experimental data. The ultraviolet-visible topotecan spectrum, recorded at moderately acidic pH, is accurately reproduced only by TD-DFT computations including solvent effects. Comparison of the experimental and calculated bands of the UV-vis spectrum at physiological pH indicates the presence of an equilibrium among different forms that is tuned by the microenvironment embedding the drug. The quantitative agreement between TD-DFT/PCM computations and experiments allows the identification of unequivocal spectroscopic signatures for different forms of topotecan.

Antineoplastic Agents↗

PubMed2005

The extent of perfusion-F18-fluorodeoxyglucose positron emission tomography mismatch determines mortality in medically treated patients with chronic ischemic left ventricular dysfunction.

OBJECTIVES: The purpose of this study was to assess the determinants of mortality in a large group of patients with ischemic cardiomyopathy who are treated medically and the impact of the extent of viable tissue on prognosis. BACKGROUND: Whether the presence of viability drives mortality in patients with ischemic cardiomyopathy who are treated medically and whether the extent of viability is important are issues that are currently unclear. METHODS: Two hundred sixty-one patients with ischemic cardiomyopathy underwent positron emission tomography (PET) for assessment of viability. Prospective follow-up was obtained. RESULTS: Ninety-four patients were revascularized and 167 were not. The cardiac death rate was significantly less in the revascularized patients as compared with medically treated patients (13% vs. 24%, p < 0.05). In the revascularized patients, there was a trend toward better survival in patients with viable myocardium as compared with nonviable myocardium (3.5-year survival, 85% and 75% respectively, p = NS). In the medically treated group, age (hazard ratio [HR] 2.1, 95% confidence interval [CI] 1.2 to 3.7), presence of left bundle branch block (HR 3.4, 95% CI 1.6 to 7.2) and extent of perfusion-metabolism mismatch on PET (HR 1.36, 95% CI 1.1 to 1.6) predicted cardiac death during a median follow-up period of 2.1 years. The risk of cardiac death was not significantly increased when the extent of mismatch was < or =20% (HR 0.97, 95% CI 0.46 to 2.05) but was significantly increased when the extent of mismatch was >20% (HR 3.21, 95% CI 1.38 to 7.49). CONCLUSIONS: Medically treated patients with ischemic cardiomyopathy and large areas of viable myocardium on PET are at high risk for cardiac death.

Aged↗

PubMed2005

X-ray absorption investigation of a unique protein domain able to bind both copper(I) and copper(II) at adjacent sites of the N-terminus of Haemophilus ducreyi Cu,Zn superoxide dismutase.

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.

Binding Sites↗

PubMed2005

Early mobilization after percutaneous catheterization and vascular closure with a novel device (Star-Close).

Arteriotomy closure devices used to obtain hemostasis at the end of cardiac catheterization represent a valid alternative to manual compression. We are the first to report on a series of 60 patients in whom a novel system was used, Star-Close, which obtains vessel closure by deployment of a clip at the puncture site. Subsequent early (2 hours) mobilization was obtained in 55 patients (92%). Unsuccessful device deployment occurred in 5 patients. We conclude that Star-Close is a novel arteriotomy closure device that, in selected patients, is safe and allows early deambulation after diagnostic cardiac catheterization.

Aged↗

PubMed2005

Purification and characterization of recombinant Caulobacter crescentus Cu,Zn superoxide dismutase.

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the metals have a crucial role in the stabilization of this class of enzymes.

Amino Acid Sequence↗

PubMed2005

Clinical, resting echo and dipyridamole stress echocardiography findings for the screening of renal transplant candidates.

BACKGROUND: Preoperative screening for coronary artery disease is recommended in high-risk renal transplant candidates. Aim of this study was to prospectively assess the value of a comprehensive risk stratification strategy including clinical, resting echo, and dipyridamole stress echo findings before renal transplantation. METHODS: The study group consisted of 71 renal transplant candidates (47 men; age 54+/-11 years) fulfilling one or more of the following high-risk clinical criteria: history of coronary artery disease, wall motion abnormalities at resting echo, dialysis dependency lasting >5 years, presence of 2 or more risk factors. Clinical history, resting echo, and dipyridamole stress echo (up to 0.84 mg over 10 min + atropine up to 1 mg) were obtained in all subjects. RESULTS: Mean number of risk factors was 2.5+/-1.0. Known coronary artery disease and diabetes were present, respectively, in 2 (3%) and 11 (15%) persons. No patient had left ventricular ejection fraction <45%. Left ventricular hypertrophy was found in 53 (74%) cases. Stress echo showed 100% safety and 97% overall feasibility. Inducible ischemia (new wall motion abnormalities) was detected in 3 (4%) subjects. During follow-up (36+/-12 months), 8 (11%) cardiac events occurred: 2 deaths, 2 myocardial infarctions, 3 coronary interventions, and 1 pulmonary edema. The perioperative period and subsequent follow-up (22+/-12 months) was uneventful among 32 patients who received renal transplantation. Four-year event-free survival was 92% in those without ischemia; it was 96% in the non-diabetic population. Diabetes (HR=4.78), age (HR=1.14), and left ventricular mass index (HR=1.02) were independent prognostic indicators among clinical and resting echo variables. The global chi-square of the statistical model was 18.8; it increased to 27.3 (+45%) after the addition of stress echo result. CONCLUSIONS: Renal transplant candidates can undergo effective stratification of risk by combining clinical, resting echo and dipyridamole stress echo findings.

Adult↗

PubMed2005

Artificial neural networks and robust Bayesian classifiers for risk stratification following uncomplicated myocardial infarction.

OBJECTIVE: To compare artificial neural networks (ANN) and robust Bayesian classifiers (RBC) in predicting outcome following acute myocardial infarction (AMI). METHODS: Clinical, exercise ECG and stress echo variables by 496 patients with AMI were used to predict the cumulative end-point of cardiac death, nonfatal reinfarction and unstable angina. Revascularized patients were censored. Short (200 days)-, medium (400 days)- and long (1000 days)-term observation intervals, including 50%, 75% and 90% of the events, respectively, were considered. At each interval, any patient was binary assigned to the "event" or "no event" class. A multilayer feedforward ANN, trained by a back propagation algorithm, was used. RBC, using the leave-one-out technique, were derived. The accuracy of both techniques was compared to the default accuracy (DA) obtained by assigning all subjects to the largest class. RESULTS: 14 death, 27 reinfarction and 29 unstable angina were observed during a mean follow-up of 24 [95% confidence interval (CI) 19 to 22] months. The accuracy of ANN and RBC and DA were 70%, 81% and 74% at short, 67%, 73% and 56% at medium and 64%, 68% and 62% at long-term follow-up. CONCLUSIONS: (1) ANN do not improve the prognostic classification of patients with uncomplicated AMI as compared to RBC. (2) In particular, short-term prognostic accuracy seems insufficient.

Algorithms↗

PubMed2005

Effect on DNA relaxation of the single Thr718Ala mutation in human topoisomerase I: a functional and molecular dynamics study.

The functional and dynamical properties of the human topoisomerase I Thr718Ala mutant have been compared to that of the wild-type enzyme using functional assays and molecular dynamics (MD) simulations. At physiological ionic strength, the cleavage and religation rates, evaluated on oligonucleotides containing the preferred topoisomerase I DNA sequence, are almost identical for the wild-type and the mutated enzymes, as is the cleavage/religation equilibrium. On the other hand, the Thr718Ala mutant shows a decreased efficiency in a DNA plasmid relaxation assay. The MD simulation, carried out on the enzyme complexed with its preferred DNA substrate, indicates that the mutant has a different dynamic behavior compared to the wild-type enzyme. Interestingly, no changes are observed in the proximity of the mutation site, whilst a different flexibility is detected in regions contacting the DNA scissile strand, such as the linker and the V-shaped alpha helices. Taken together, the functional and simulation results indicate a direct communication between the mutation site and regions located relatively far away, such as the linker domain, that with their altered flexibility confer a reduced DNA relaxation efficiency. These results provide evidence that the comprehension of the topoisomerase I dynamical properties are an important element in the understanding of its complex catalytic cycle.

Alanine↗

PubMed2005

Predictive value of associations between carotid and coronary artery disease in patients with acute coronary syndromes.

The present study evaluated the association among sonographic characteristics of carotid plaques, angiographic morphology of coronary lesions, and outcomes of patients who had acute coronary syndromes. In treatment-stratified Cox's proportional hazards regression analysis, hard carotid plaques and coronary lesions of complex morphology were found to be independent predictors of future cardiac events.

Acute Disease↗

PubMed2005

Echocardiography for risk stratification of myocardial infarction in the reperfusion era.

The prediction of functional recovery or ventricular remodeling plays a central role in the prognostic assessment of acute myocardial infarction (MI) in the reperfusion era. This article reviews the advances of echocardiographic techniques, focusing on the role of low-dose dobutamine stress echocardiography, contrast echocardiography, transthoracic Doppler of the left anterior descending coronary artery, and transmitral Doppler in detecting myocardial viability and predicting outcome after an acute MI. Moreover, the definition of risk for further ischemic events is another major objective in conservatively managed patients post infarction. As such, the prognostic and economic implication of stress echocardiography compared with that of traditional exercise electrocardiography was considered.

Coronary Vessels↗

PubMed2005

Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family.

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Amino Acid Motifs↗

PubMed2004

Impact of carotid arterial narrowing on outcomes of patients with acute coronary syndromes.

The present prospective observational study was conducted in 323 consecutive patients with acute coronary syndrome, 101 (31%) of whom were diagnosed with carotid arterial narrowing by ultrasonography. At follow-up, patients with carotid disease had a greater incidence of cardiovascular events than did those with normal carotid arteries. Outcomes of patients with concomitant carotid disease who underwent cardiac revascularization were similar to those who received conservative treatment.

Aged↗

PubMed2004

Molecular dynamics simulation of the C-terminal sterile alpha-motif domain of human p73alpha: evidence of a dynamical relationship between helices 3 and 5.

We used molecular dynamics simulation to evaluate the association properties of C-terminal sterile alpha-motif (SAM) domain of human p73alpha. To test the dimerization propensity of this structure we carried out four simulations: EphB2 X-ray dimer, p73 modeled dimer, p73 NMR monomer, and p73 modeled monomer with an elongated helix 5. The results show a direct interaction between helix 5 and helix 3 since a conformational collapse of helix 3 is observed when dimer contact and/or an elongation of helix 5 is introduced by modeling in p73 SAM domain. On the basis of these results we suggest that the recognition properties of the SAM domains may be modulated by the conformational state of helix 5.

Amino Acid Motifs↗

PubMed2004

Effect of pharmacological wash-out in patients undergoing exercise testing after acute myocardial infarction.

STUDY OBJECTIVES: Pharmacological therapy can reduce diagnostic and prognostic accuracy of exercise stress testing. However, the risk of withdrawing drugs early after myocardial infarction (MI) has not been established. We assessed safety and clinical implications of drug withdrawal in patients undergoing stress testing after uncomplicated MI. METHODS: A total of 362 MI patients underwent ECG Holter recording before and after withdrawing beta-blockers, calcium-antagonists and nitrates. QRS (QRS/h) and ventricular premature beats (VPB/h) count per hour, repetitive ventricular arrhythmias, ST segment changes and patient complaints were evaluated for reproducibility using kappa statistics and Bland-Altman method. RESULTS: No major complications occurred. Forty-three patients complained of >1 symptom on and 37 off therapy. QRS/h and VPB/h count were significantly (p<0.0001) higher off therapy but correlated with the corresponding values on therapy. A mean heart rate increase of 8 beats/min (agreement range -8 to +14 beats/min) and a five-fold increase in VPB/h (agreement range -141 to +151) were observed after withdrawing therapy. Repetitive ventricular arrhythmias and ST changes were also more frequent off therapy but intra-patient reproducibility was poor: kappa 0.12 (95% confidence interval (CI) -0.01 to 0.25) for arrhythmias, -0.02 (95% CI -0.46 to 0.39) for ST depression and -0.01 (95% CI -0.66 to 0.64) for ST elevation. CONCLUSIONS: The withdrawal of therapy is well tolerated soon after uncomplicated MI; however, a generic but not individual risk of ventricular arrhythmias and/or transient myocardial ischemia has to be taken into account.

Adrenergic beta-Antagonists↗

PubMed2003

Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I.

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.

Alanine↗

PubMed2003

Two distinct calcium-calmodulin interactions with N-terminal regions of the olfactory and rod cyclic nucleotide-gated channels characterized by NMR spectroscopy.

The interactions of calcium-calmodulin with two fragments of the N-terminal domains of the olfactory alpha-subunit and rod beta-subunit cyclic nucleotide-gated channels have been investigated using nuclear magnetic resonance spectroscopy. The results indicate that in the two cases both the N-terminal and the C-terminal calmodulin lobes are involved in the interaction. The olfactory cyclic nucleotide-gated channel segment forms a 1:1 complex with calmodulin, whereas the rod fragment forms a 2:1 complex. The correlation times of the two complexes, as estimated by (15)N relaxation studies, are compatible with the observed stoichiometries. These results indicate differences in the mode of action by which calmodulin modulates the activity of both channels, and suggest either that the rod channel is modulated through a simultaneous interaction of two beta-subunits with calmodulin or that other regions of the N-terminus are necessarily implicated in the binding.

Amino Acid Sequence↗