Progenitor cell mobilisation in patients with acute and chronic coronary artery disease.
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Acute Disease↗
Biomedical subjects
G Barbato
Publications and source records attributed to G Barbato.
Dynamics of electroencephalographic slow wave activity and body temperature during monophasic and biphasic human sleep.
Dynamics of electroencephalographic (EEG) slow wave activity (0.5-4.5 Hz) and body temperature, as estimates, respectively, of the process S and process C, regulating sleep and waking alternate occurrence, were measured during monophasic and biphasic sleep patterns that occurred spontaneously in a 35-year-old woman who lived for 105 days in a winter-type photoperiod (10-14 h light-dark). In monophasic nights, rate of EEG synchronization showed a decreasing trend across the first three non-rapid eye movement (NREM) periods. In biphasic nights, rate of EEG synchronization increased during the third NREM period which precedes the nocturnal awakening. Temperature cycle was not different between biphasic and monophasic nights. Those results confirm that EEG dynamics reflects homeostatic sleep regulatory mechanism, and suggest that the period of prolonged wakefulness in the middle of biphasic night is pre-programmed.
Adult↗
Sensitivity enhancement of a two-dimensional experiment for the measurement of heteronuclear long-range coupling constants, by a new scheme of coherence selection by gradients.
In this work we present a new pulse sequence for the measurement of long-range heteronuclear coupling constants in which the optimization of coherence selection by pulsed field gradients offers a net increase in sensitivity. This type of experiments is extremely valuable for conformational studies of molecules in natural abundance and in this context the use of gradients is essential for an efficient suppression of (12)C bound proton signals. A comparative analysis of the different gradient schemes available is presented with a conclusive elucidation of the relative sensitivities. Our gradient scheme could be advantageous as a building block for other related experiments.
Journal Article↗
Improved sensitivity in indirect monitoring of chemical shifts of proton-heteronuclear spin pairs (1H-13C and 1H-15N) in 3D and 4D NMR spectroscopy.
In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (13C or 15N) but also proton (1H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying 1H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of 1H signals, yielding improved sensitivity. The new method applies to spin pairs like 1H-15N, as in amide groups, or 1H-13C, as in methine groups of alpha or aromatic systems.
Carbon Isotopes↗
Measurement of homonuclear three-bond J(H(N)Halpha) coupling constants in unlabeled peptides complexed with labeled proteins: application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV).
A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H(N)Halpha) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the 'quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV).
Algorithms↗
Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain.
Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His-Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His-Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.
Aminobutyrates↗
Diurnal variation in spontaneous eye-blink rate.
The daily pattern of spontaneous eye-blink rate (BR), a non-invasive peripheral measure of central dopamine activity, was investigated in 24 healthy subjects. The spontaneous eye-blink rate showed a stable pattern in morning, midday and afternoon hours. A significant increase was found at the evening time point (20.30 h). The finding is suggestive of a late evening increase of central dopamine activity. An increased level of subjective sleepiness was also found at the same evening point, at a time corresponding to the 'evening wake maintenance zone' or the 'forbidden zone for sleep'. A possible hypothesis is that the 'forbidden zone for sleep' may reflect a dopamine-mediated activation that counteracts a rising sleep drive. The role of diurnal variation of dopamine function should be considered both in the choice of the drug treatment regimen, and in the evaluation of biological and neuropsychological parameters.
Adolescent↗
The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.
The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.
Amino Acid Sequence↗
Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies.
The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.
Amino Acid Sequence↗
Selective correlation of amide groups to glycine alpha protons and of arginine guanidine groups to delta protons in proteins by multiple quantum spectroscopy.
A new 3D pulse sequence correlates backbone amide proton and nitrogen with alpha proton resonances selectively for glycine residues in a fully doubly labeled (15N, 13C) protein. The excitation of multiple quantum coherences provides optimized resolution and sensitivity. Degenerate alpha proton groups can be promptly recognized. Correlation of guanidine NH groups to delta protons of arginine side chains is also obtained.
Amides↗
Extrapyramidal syndromes in neuroleptic-treated patients: prevalence, risk factors, and association with tardive dyskinesia.
Prevalence and risk factors for extrapyramidal syndromes (EPS) were investigated in a sample of 1,559 patients. The overall prevalence of EPS was 29.4% (N = 458). Among the EPS-diagnosed patients, parkinsonism as assessed by the presence of core parkinsonian symptoms (rigidity, tremor, bradykinesia) was present in 65.9% of patients (N = 302), akathisia in 31.8% (N = 145), and acute dystonia in 2.1% (N = 10). Old age and long-term neuroleptic drug (NL) treatment were significantly associated with EPS in both the univariate and the multivariate analyses, whereas no relationship was observed with average NL daily doses and current NL treatment. EPS was diagnosed in 50.2% of 285 patients with persistent tardive dyskinesia (TD). Distribution of EPS in patients with TD showed that tremor and akathisia were more frequent in peripheral TD cases than in orofacial TD cases. Furthermore, there was a stronger association of NL-induced parkinsonism with peripheral TD than with orofacial TD. This study suggests that the association between EPS and TD may be limited to specific subtypes of TD. Peripheral TD showed a higher association with parkinsonism and with akathisia, suggesting that these symptoms may share a common pathophysiology.
Adolescent↗
The metal binding site of the hepatitis C virus NS3 protease. A spectroscopic investigation.
The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines (Cys97, Cys99, and Cys145) and one histidine residue (His149), which is postulated to coordinate the metal through a water molecule. In this article, we present an analysis of the role of metal coordination with respect to enzyme activity and folding. Using NMR spectroscopy, the resonances of His149 were assigned based on their isotropic shift in a Co(II)-substituted protein. Data obtained with 15N-labeled NS3 protease were compatible with the involvement of the delta-N of His149 in metal coordination. pH titration experiments showed that the cooperative association of at least two protons is required in the protonation process of His149. Changes in the NMR signals of this residue between pH 7 and 5 are interpreted as evidence for a structural change at the metal binding site, which switches from a "closed" to an "open" conformation. Site-directed mutagenesis of His149 has shown the importance of this residue in the metal incorporation pathway and for achieving an active fold. The metal coordination of the protease was also investigated by circular dichroism and electronic absorption spectroscopies using a Co(II)-substituted enzyme. We show evidence for rearrangements of the metal coordination geometry induced by complex formation with an NS4A peptide cofactor. No such changes were observed upon binding to a substrate peptide. Also, CN- and N3- induced Co(II) ligand field perturbations, which went along with an 1.5-fold enhancement of protease activity.
Amino Acid Substitution↗
Homeostatic regulation of REM sleep in humans during extended sleep.
Benington and Heller (1994) recently proposed a sleep-dependent model for the homeostatic control of REM sleep in which the amount of REM sleep propensity discharged in each bout of REM sleep affects the timing of the subsequent REM episode. Consistent with their hypothesis, they reported that in rats the duration of a REM episode was positively correlated with the duration of the succeeding NREM episode and not with the duration of the preceding NREM episode. To assess this hypothesis in humans, we used 308 sleep records from 11 subjects who remained at bedrest in the dark and slept ad libitum during 14-hour periods each night for 4 weeks. The timing of the onset of the first REM episode of the long night was linked to the timing of sleep onset. NREM-REM cycle duration decreased progressively throughout the night as a result of a progressive decrease in duration of the NREM component. Durations of REM sleep episodes correlated significantly with durations of subsequent NREM episodes in three out of the eight rank cycles analyzed (p < .0031, Bonferroni corrected); positive correlation coefficients were found for all the remaining cycles, but were not statistically significant when the conservative Bonferroni correction of the alpha level was applied. With the exception of the first sleep cycle, durations of REM sleep episodes did not correlate with durations of preceding NREM sleep episodes. According to the present results, the amount of REM sleep in one episode controls the time of occurrence of the next REM episode when the impact of other possible regulating factors are at a low level. We hypothesize that the extended dark/rest period, by increasing the time window allowed for sleep, provided a condition under which the systems governing REM sleep expression were free of the masking imposed by the conventional 16 hours light/8 hours dark schedule that consolidates and compresses sleep.
Adult↗
Resonance assignment and secondary structure determination and stability of the recombinant human uteroglobin with heteronuclear multidimensional NMR.
Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-dependent, 17 kDa homodimeric, secretory protein with potent anti-inflammatory/immunomodulatory properties. However, the exact physiological role still remains to be determined. It has been hypothesised that its activity is exerted through the binding of a specific target represented by a small molecule (still unknown), and that the binding is regulated by the formation/disruption of two cysteine bonds. The binding properties of the reduced UG have been proved in vitro for several different molecules, but no in vivo data are available to date. However, binding has been observed between reduced rabbit UG and a protein of an apparent molecular mass of 90 kDa and, more recently, we found an h-UG-binding protein
Animals↗
Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field.
The effects of intermittent exposure (2 h on/22 h off) to a 200 microT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 microT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs.
Animals↗
High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif.
We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the alpha-helical portion of a 26-residue 'consensus' peptide (CP1) belonging to the Cys(2)-Hys2 zinc-finger family. The result was shown to be a highly homogeneous alpha-helical library (Bianchi et al., 1995). The structures of the parent compound (CPI) and of a representative member (CPlm) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.
Magnetic Resonance Spectroscopy↗
Effects of sleep deprivation on spontaneous eye blink rate and alpha EEG power.
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Adult↗
A conformationally homogeneous combinatorial peptide library.
In search for a rational way to convert the information encoded in peptide structures into peptidomimetics, major progress could be made by coupling the power of selection methods, now enormously increased in number as a result of the development of combinatorial peptide libraries, with the rational design of structure-inducing templates for the selectable sequences. The availability of libraries of peptides with predetermined structure would enable selection-driven peptidomimetic design, whereby a conformational model for the peptide pharmacophore would be directly derived from the screening, allowing the design of a suitable non-peptidic scaffold to replace the peptide backbone. We describe here the first example of a conformationally homogeneous combinatorial peptide library, which yields ligands with the expected structure upon selection. The library was built by randomising five positions in the alpha-helical portion of a 26 amino acid Cys2His2 consensus "zinc-finger" motif. Since in zinc-fingers metal coordination and folding are coupled, in our library metal-dependent binding represents a built-in control against the selection of structurally undefined sequences. The alpha-helical library was produced as both fusion with the pVIII protein of filamentous phage and soluble peptides by chemical synthesis, the latter enabling the expansion of the selectable repertoire by the inclusion of non-coded amino acids. The two libraries were independently screened with the same receptor (a monoclonal IgA reactive against the lipopolysaccharide of the human pathogen Shigella flexneri), yielding a very similar consensus. In particular, the peptides defined by both methods showed very strong, zinc-dependent binding to the IgA. The geometrical arrangement of the side-chains of the selected peptide pharmacophore was shown by circular dichroism, Co(II)-complex absorption and high-resolution NMR to be structurally invariant with respect to the parent zinc-finger.
Amino Acid Sequence↗