Biomedical subjects

A Desideri

Publications and source records attributed to A Desideri.

At least 109 records · Page 6Linked to original sources

PubMed1996

Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: a site-directed mutagenesis approach.

The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double (Lys 120-->Leu-Lys 134-->Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.

Animals↗

PubMed1996

Crystallization and preliminary X-ray analysis of the monomeric Cu,Zn superoxide dismutase from Escherichia coli.

The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2(1), with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, beta = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.

Amino Acid Sequence↗

PubMed1996

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.

Animals↗

PubMed1996

Effect of Lys-->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium.

The thermal stability of two single (K3R, K67R) and one double (K3R-K67R) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied to test Lys --> Arg substitution as an 'electrostatically conservative' strategy to increase protein stability. The K3R mutant displays an increased thermostability with respect to the wild-type enzyme, whilst a decreased stability was observed in the case of the K67R and K3R-K67R mutants. Concentration dependence of the apparent inactivation constant (kapp) of the latter mutants, as compared to that of the wild type enzyme and K3R mutant, indicates that their higher sensitivity to heat inactivation is due to a perturbation of the dimer association. These results are confirmed also by fluorescence anisotropy measurements of the internal probe Tyr149. The possible role of Arg67 in perturbing the dimer dissociation equilibrium toward the monomeric form is discussed.

Animals↗

PubMed1996

Left ventricular diastolic function in liver cirrhosis.

BACKGROUND: Left ventricular systolic abnormalities have been reported in liver cirrhosis (LC). Diastolic function in cirrhotics, on the contrary, does not seem to have been studied so far. METHODS: Diastolic function was evaluated in 42 cirrhotic patients and in 16 controls by means of Doppler echocardiography. RESULTS: Compared with the controls, cirrhotics had increased left ventricular end-diastolic and left atrial volume, stroke volume, late diastolic flow velocity (peak A) (71 + or - 17 cm/sec versus 56 +/- 18; p <0.01), time from onset of mitral inflow to the early peak (time E) (86 + or - 11 msec versus 72 +/- 14; p < 0.003), and deceleration time (DT) (194 +/- 40 msec versus 159 +/- 27; p < 0.001) and decreased ratio of peak E to peak A filling velocities (1.02 +/ - 0.35 versus 1.22 +/- 0.25; p < 0.02). Patients with tense ascites had a higher E/A ratio (p < 0.03) and a shorter DT (p < 0.03) than patients with mild or no ascites. CONCLUSIONS: The impaired left ventricular relaxation in the presence of high stroke volume suggests a myocardial involvement in LC. The pseudo normalization of the E/A ratio and DT in patients with tense ascites could reflect loading conditions masking the relaxation abnormality.

Ascites↗

PubMed1996

[Combined use of bicycle ergometry test and echo-dipyridamole in stratification of risk after thrombolysed myocardial infarct].

AIM: Verify the value of the combined use of Dipyridamole Stress Echocardiography (DSE) and Exercise Testing (ET) for predicting cardiac events after Thrombolysed Myocardial Infarction (TMI). STUDY DESIGN: Prospective, with a medium follow-up of 221 days (range 30-446). CLINICAL SETTINGS: Coronary Care Unit with Cardiology Service. PATIENTS: 32 consecutive patients, 24 men and 8 women, mean age 61 years (range 39-72) with acute myocardial infarction. INTERVENTIONS: Systemic thrombolysis with PTPA. DSE and ET in therapeutic wash out 12 +/- 3 days after admission to the Coronary Care Unit. RESULTS: 9 patients had positive ET, 18 patients had negative ET, 5 patients did not perform ET. 19 patients had positive DSE, 13 patients had negative DSE. Patients with positive DSE had in 6 cases (group 1) WMA remote from the infarction area (WMAR), in 13 cases (group 2) WMA appeared in the infarct related area. Cardiac events occurred in 15 patients: 1 cardiovascular death, 1 myocardial re-infarction, 13 cases of unstable angina. 2 patients with negative DSE had cardiac events. All patients in group 1 had events, 1 patient with a previous positive ET, 3 patients with previous negative ET. 2 patients in this group didn't perform ET. 7 patients in group 2 had cardiac events, in all cases with a previous positive ET. 1 single patient in this group with negative ET had events. CONCLUSIONS: 1) Patients without WMA at DSE are at low risk for cardiac events after thrombolysed myocardial infarction. 2) Presence of WMAR appears to be associated with a higher risk, whatever the result of ET may be. 3) Patients without WMAR can be further stratified by ET in therapeutic wash out. High risk patients seem to be those with positive DSE and positive ET. 4) Combined use of DSE and ET seem to allow optimal risk stratification after thrombolysed myocardial infarction.

Adult↗

PubMed1995

Low-temperature optical spectroscopy of cobalt in Cu,Co superoxide dismutase: a structural dynamics study of the solvent-unaccessible metal site.

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectivity replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N3(-)-Cu(II), Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(II) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm-1; the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Addition of N3- to the Cu(II),Co(II) SOD introduces slight changes in all the Co(II) visible bands, indicating the occurrence of minor perturbations of the structural cobalt site upon anion binding to the catalytic copper site. Analysis of the spectra in the Cu(I),Co(II) and E,Co(II) enzymes indicates that the His61 imidazolate bridge is released from the copper upon reduction. This is also confirmed by the analysis of the zeroth, first, and second moments of the various bands in the derivatives. The cobalt site is characterized by a harmonic dynamics, at variance with what observed in the solvent accessible copper site [Cupane, A., Leone, M., Militello, V., Stroppolo, M. E., Polticelli, F., & Desideri, A. (1994) Biochemistry 33, 15103-15109]. The degree of local microheterogeneity at the cobalt site is smaller than that observed for the copper site and increases in the order N3(-)-Cu(II),Co(II) approximately Cu(II),Co(II) < Cu(I),Co(II) < E,Co(II) indicating a different local packing and the presence of different constraints on the cobalt site in the four derivatives. The different dynamic behavior with respect to the catalytic, solvent-accessible, copper site is discussed.

Animals↗

PubMed1995

Metal uptake of recombinant cambialistic superoxide dismutase from Propionibacterium shermanii is affected by growth conditions of host Escherichia coli cells.

We constructed the complete nucleotide sequence coding for the cambialistic superoxide dismutase from Propionibacterium shermanii by ligation of a synthetic linker to a polymerase chain reaction amplification product obtained using degenerate primers. We set up an expression system yielding large amounts of recombinant superoxide dismutase in the cytoplasm of Escherichia coli and purified the enzyme from cells grown in a complex medium. The physicochemical properties of the recombinant enzyme were identical to those of the natural protein. Under anaerobic conditions the enzyme produced in an iron-supplemented medium incorporated iron as metal cofactor, while the enzyme purified from cells grown under aerobic conditions contained a variable amount of iron and manganese depending on metal availability. Functional equivalence of the two metals in this superoxide dismutase variant was indicated by independence of enzyme activity from Fe/Mn ratio.

Amino Acid Sequence↗

PubMed1995

Identification of the residues responsible for the alkaline inhibition of the activity of Cu,Zn superoxide dismutase: a study of native and chemically modified enzymes.

The pH dependence of the activity of Cu,Zn superoxide dismutases from bovine erythrocytes and shark liver was studied by pulse radiolysis in both the native enzymes and those chemically modified at lysine side chains. The study was aimed at identifying the residues responsible for the activity decrease at pH > 9, observed in all native Cu,Zn superoxide dismutases, and is based on the Lys-->Arg substitution present in the shark protein at position 134, which has been established to be critical for the catalytic efficiency of the enzyme. Both native enzymes display a pH dependence that can be deconvoluted by three deprotonation equilibria, at pH 9-9.5 (pK1), at pH 10.2 (pK2), and at pH 11.5 (pK3). pK1 is lacking in both the modified enzymes and thus can be assigned to activity-linked lysine residues. pK2 is clearly dominated by Arg134 in the modified shark enzyme and can be assigned to surface arginine residues. pK3 is shared by all four enzyme forms and is likely to be due to the invariant Arg141.

Amino Acid Sequence↗

PubMed1995

Evidence for breaking of the active site dimetal cluster in Cu,Co superoxide dismutase upon copper reduction: a polarized absorption spectra study.

The polarized absorption spectra of the cobalt chromophore in orthorhombic crystals of bovine Cu,Co superoxide dismutase (SOD), bearing the copper ion in both the oxidized and the reduced states, are reported together with the calculated isotropic spectrum. All the absorption bands are polarized and a spectral shift, from 598 nm to 588 nm, is observed in only one of them upon copper reduction. This shift, previously described in solution studies, is unequivocally assigned to the detachment of the copper side from the imidazolate bridging copper and cobalt ions in the oxidized catalytic center. At variance with a recent X-ray diffraction investigation on Cu(I),Zn SOD, this result indicates that reduction of copper in Cu,Co SOD is associated to the breaking of the dimetal cluster also in the crystalline state.

Animals↗

PubMed1995

A single mutation (Thr72-->Ile) at the subunit interface is crucial for the functional properties of the homodimeric co-operative haemoglobin from Scapharca inaequivalvis.

The in vivo expression and the functional and spectroscopic properties are reported for a mutant of the homodimeric haemoglobin of the mollusc Scapharca inaequivalvis (HbI), where residue threonine 72 (position 9 in the E helix) at the subunit interface has been substituted by isoleucine. The aim of this study is to test the hypothesis that increasing the hydrophobicity character of the subunit interface may modulate oxygen affinity and co-operativity of this haemoglobin. In fact, X-ray crystal structure studies have shown that the subunit interface, formed by the E and F helices of the two chains, changes its character from hydrophilic to hydrophobic upon oxygenation. This is primarily due to extrusion of Phe97 side-chain from the haem pocket toward the interface, which disrupts a network of ordered water molecules and results in close van der Waals contacts between Phe97 and Thr72 of the partner subunit. Thr72-->Ile HbI was expressed in E. coli after mutation of HbI-DNA and it displays a approximately 40-fold enhancement of oxygen affinity and a marked reduction of co-operativity in oxygen binding, with respect to native HbI. These functional properties and the kinetics of oxygen dissociation and carbon monoxide combination rates, as well as data from EPR and circular dichroism spectroscopy, indicate that indeed the increase of the hydrophobicity at the interface upon mutation stabilizes the "high affinity" conformation of the protein, suggesting that extrusion of Phe97 toward the interface should be facilitated even in the unliganded form.

Animals↗

PubMed1995

Modulation of the catalytic rate of Cu,Zn superoxide dismutase in single and double mutants of conserved positively and negatively charged residues.

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength.

Animals↗

PubMed1995

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.

Animals↗

PubMed1995

A two-dimensional NMR study of bovine Cu, Co superoxide dismutase. Further assignments in the region surrounding the active site.

Bovine Cu, Co superoxide dismutase has been investigated by two-dimensional NMR with regard to the resonances arising from protons that surround the copper site. These protons have magnetic properties that are intermediate between those belonging to residues coordinated to the paramagnetic metal center and the diamagnetic residues. Nuclear Overhauser enhancement spectroscopy spectra have been recorded by using a new procedure and with different mixing times and different spectral widths, in order to observed dipolar connectivities between isotropically shifted and diamagnetic resonances. Scalar-correlated spectra were obtained with both correlation spectroscopy and total-correlation spectroscopy experiments. The original X-ray coordinates of the Cu, Co enzyme were used in order to obtain the appropriate interproton distances. The data allowed us to assign more than 20 new resonances to protons which cover a wide region around the copper ion, including two protons belonging to the catalytically important Arg141 residue. The results represent significant progress in the effort to elucidate the three-dimensional features of the region surrounding the active site of Cu, Zn superoxide dismutase in solution and a tool for a deeper investigation of the reaction mechanism of the enzyme.

Animals↗

PubMed1995

Formate binding to ferric wild type and mutant myoglobins thermodynamic and X-ray crystallographic study.

The X-ray crystal structure of the formate derivative of ferric loggerhead sea turtle (Caretta caretta) Mb has been determined at 2.0 A resolution (R = 0.164) by difference Fourier techniques. Formate, sitting in the central part of the heme distal site, is coordinated to the heme iron as unidentate ligand, through the O1 oxygen atom, and is hydrogen bonded to the distal His64(E7) NE2 atom through O2. Thermodynamics for formate binding to ferric loggerhead sea turtle Mb, sperm whale Mb, Aplysia limacina Mb, as well as to the VR and VRS mutants of sperm whale Mb were obtained between pH 4.5 and 8.5, at 20.0 degrees C. These results, representing the first structure of a ferric hemoprotein:formate complex solved by X-ray crystallography, outline the role of amino acid residues at positions E7, F8 and E10 in modulating ligand binding properties of oxygen carrying proteins.

Animals↗

PubMed1995

Dynamics of hydrogen atoms in superoxide dismutase by quasielastic neutron scattering.

The low energy dynamic of the enzyme Cu,Zn superoxide dismutase have been investigated by means of quasielastic neutron scattering in the temperature range 4-320 K. Below 200 K the scattering is purely elastic, while above this temperature a pronounced decrease in the elastic intensity is observed, together with the onset of a small quasielastic component. This behavior is similar to that previously observed in other more flexible globular proteins, and can be attributed to transitions between slightly different conformational substates of the protein tertiary structure. The presence of only a small quasielastic component, whose intensity is < or = 25% of the total spectrum, is related to the high structural rigidity of this protein.

Animals↗

PubMed1995

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves.

The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.

Amino Acid Sequence↗

PubMed1995

[Revision and optimization of processes: a fundamental timing for adequate use of the resources and technological innovation. An example of intervention in the cardiology field and considerations on "total quality" in medicine].

UNLABELLED: The Cardiology Unit of the Este General Hospital began its activity in 1988. We soon identified a mismatch between a good, up-to-date diagnostic instrumentation with growing customers' demand and an inadequate utilization of the instruments. Waiting lists were getting longer, customers were not satisfied, no-shows at the appointments increased and we had a progressive loss in image. We therefore decided to intervene on our processes, starting the project "TOTAL QUALITY IN CARDIOLOGY". We focused our attention on two main fields, namely (1) electrocardiography, (2) other diagnostic techniques, separately analyzed because of important differences. Point (1) is basically worked out by paramedical personnel, in high numbers and with stable demand, while point (2) is determinantly linked to medical activity, although with concomitant need for paramedical support. The figures are lower for point (2) but are steadily growing. In the two operating fields we further identified two separate adverse effects: 1). ELECTROCARDIOGRAPHIC EXAMINATIONS (ECG) ARE TOO TIME CONSUMING, 2) THE NUMBER OF DIAGNOSTIC PROCEDURES IS TOO LOW FOR THE INSTRUMENTS AVAILABLE. We used preliminary analysis with process flow diagrams and our interventional methods were policy deployment and daily routine work. ELECTROCARDIOGRAPHIC EXAMINATIONS ARE TOO TIME CONSUMING. From cause-effect diagram for cause classification and subsequent Pareto analysis we identified two groups of main causes: 1. the paramedical-patient team is not able to optimise usage of the instrument; in particular, total time for undressing of the patient, lead attachment, dressing of the patient, change in ECG conductivity cream and, if necessary, repetition of ECG for mistakes in procedure is much longer than operative time of the instrument; 2. the necessary copy of the ECG done by the instrument was too time consuming (2') as compared to total procedure time. Implementation plans have been as follows: activity was concentrated in one single room at constant temperature (20 degrees); we augmented the number of dressing rooms and nurses (from 1 to 2 unit); we substituted the ECG conductivity cream with a water-alcoholic solution and the copy with a photocopy. RESULTS: decrease in mean time for ECG from 6'52" to 3'25" (for example: total ECGs 1992: 14,827, total spared time: 852 working hours); reductions in dead times; capability to cope rapidly with high demand; consequent possibility to utilise paramedical personnel for other activities; reduction in copy costs from 156 to 50 Lit each (total reduction 1,571,662 Lit). EXPECTATIONS. further revision of the procedures to keep pace with new electrocardiographic instruments and to achieve shorter operative times.

Cardiology↗