Dynamics of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

A significant determinant for the broad substrate specificity of the metallo-beta-lactamases from Bacteroides fragilis and other similar organisms is the presence of a plastic substrate binding site that is nevertheless capable of tight substrate binding in the Michaelis complex. To achieve these two competing ends, the molecule apparently employs a flexible flap that closes over the active site in the presence of substrate. These characteristics imply that dynamic changes are an important component of the mechanism of action of these enzymes. The backbone and tryptophan side chain dynamics of the metallo-beta-lactamase from B. fragilis have been examined using (15)N NMR relaxation measurements. Two states of the protein were examined, in the presence and absence of a tight-binding inhibitor. Relaxation measurements were analyzed by the model-free method. Overall, the metallo-beta-lactamase molecule is rigid and shows little flexibility except in loops. The flexibility of the loop that covers the active site is not unusually great as compared to the other loops of the protein. Local motion on a picosecond time scale was found to be very similar throughout the protein in the presence and absence of the inhibitor, but a significant difference was observed in the motions on a nanosecond time scale (tau(e)). Large-amplitude motions with a time constant of about 1.3 ns were observed for the flexible flap region (residues 45-55) in the absence of the inhibitor. These motions were completely damped out in the presence of the inhibitor. In addition, the motion of a tryptophan side chain at the tip of the beta-hairpin of the flap shows a very significant difference in motion on the ps time scale. These results indicate that the motions of the polypeptide chain in the flap region can be invoked to explain both the wide substrate specificity (the free form has considerable amplitude of motion in this region) and the catalytic efficiency of the metallo-beta-lactamase (the motions are damped out when the inhibitor and by implication a substrate binds in the active site).     

Spindle activity during sleep in subjects with a deficit in pallesthetic sensibility

Sleep spindle activity was studied on four subjects affected by pallesthesic deficit due to injury the posterior funiculi of the cord. The spindle activity was studied as density (number of spindles min.), duration and percent of sleep time utilized in spindle activity. The polygraph sleep records included EOG, EMG and 6 monopolar EEG recordings, 3 for each side, on the frontal, parietal and occipital regions. The records showed a spindle activity which was similar in different subjects and that was significantly higher than the physiological values. In fact, the spindle density was about 250%, the duration was about 130% and the spindle percent was about 280%, with respect to the physiological values assumed to be 100%.     

NMR characterization of the metallo-beta-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: role of an active-site loop.

Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-beta-lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular beta-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'-mercaptopropanoyl]-4-(S)-carboxy-5, 5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the beta-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the beta-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-beta-lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.     

Electroencephalographic characteristics of sleep in subjects with paleo and neocerebellar lesions

The research was performed in order to study: 1) paleo and neocerebellar contributions in the sleep organization and 2) the electrical sleep activities at different time intervals during the functional compensation which follows the cerebellar lesion. Polygraphic sleep records (EEG, EMG, EOG) were performed on four subjects with surgical lesions more than 6 months old in cerebellar cortex (two subjects in paleo and two in neocerebellum). Another subject was studied before a surgical paleocerebellar lesion and at different time intervals after that (8th, 30th, 60th, and 90th day). Paleo and neocerebellar lesions showed different sleep abnormalities. The former induced both quantitative and qualitative alterations in the cyclic sleep organization, the latter did not show significant alterations in this organization but rather in transition between sleeping and waking and in sleep maintenance. The acute paleocerebellar lesion showed at the 8th and 30th day a strong reduction of the synchronized sleep (SS) and an increase of the desynchronized one (DS). In the successive records, 30th and 90th day, the SS/DS ratio increased to the values observed in the chronic paleocerebellar lesioned subjects.     

Computational study of the risk of restenosis in coronary bypasses

Biomechanics and Modeling in Mechanobiology - Coronary artery disease, caused by the buildup of atherosclerotic plaques in the coronary vessel wall, is one of the leading causes of death in the...     

Numerical modeling of hemodynamics scenarios of patient-specific coronary artery bypass grafts

A fast computational framework is devised to the study of several configurations of patient-specific coronary artery bypass grafts. This is especially useful to perform a sensitivity analysis of the hemodynamics for different flow conditions occurring in native coronary arteries and bypass grafts, the investigation of the progression of the coronary artery disease and the choice of the most appropriate surgical procedure. A complete pipeline, from the acquisition of patient-specific medical images to fast parameterized computational simulations, is proposed. Complex surgical configurations employed in the clinical practice, such as Y-grafts and sequential grafts, are studied. A virtual surgery platform based on model reduction of unsteady Navier–Stokes equations for blood dynamics is proposed to carry out sensitivity analyses in a very rapid and reliable way. A specialized geometrical parameterization is employed to compare the effect of stenosis and anastomosis variation on the outcome of the surgery in several relevant cases.