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Biomedical subjects

A Bellelli

Publications and source records attributed to A Bellelli.

At least 19 recordsLinked to original sources
PubMed2006

Magnetic resonance imaging of posterior cruciate ligament injuries: a new classification of traumatic tears.

PURPOSE: The aim of our study was to propose a new classification of acute posterior ligament (PCL) injuries based on magnetic resonance imaging (MRI). MATERIALS AND METHODS: Using MRI, we reviewed 220 patients presenting an acute posterior ligament injury. The MRI exam was performed on a 0.2-Tesla (T) magnet and a 1.5-T magnet using T1-weighted spin echo (SE), T2-weighted SE and fat-suppression scans [short-tau inversion recovery (STIR)] in axial, sagittal and coronal planes. In no case was paramagnetic contrast agent injected. RESULTS: Twenty-five per cent of patients were identified as having type I lesions and another 30% as having type II lesions according to the Gross classification. Out of 77 patients, 35% of the whole sample, 37 had a tear of the posteromedial fascicle (type II lesion), and the remaining 40 had anterolateral fascicle tears (type III). In 10% of cases, the ligaments appeared completely interrupted, and these were classified as type IV lesions. CONCLUSIONS: T1-weighted SE sequences need to be integrated with T2-weighted and STIR sequences to detect the real extent of the remaining fascicle. The MRI exam could thus be able to provide a qualitative evaluation of PCL injuries and establish how the injury compromises joint stability.

Adolescent↗
PubMed2006

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

Animals↗
PubMed2002

MR evaluation of anterior cruciate ligament reconstruction with quadriceps tendon. Preliminary experience.

PURPOSE: Anterior cruciate ligament reconstructions are becoming increasingly frequent, and MRI has been shown to be the best imaging modality for the non-invasive assessment of surgical outcome. Use of the quadriceps tendon as a biological replacement for injured cruciate ligament is a recent innovation. This study evaluated by MRI the results of anterior cruciate reconstruction in 27 consecutive patients who underwent arthroscopic reconstruction with homologous quadriceps tendon. MATERIAL AND METHODS: MRI was carried out on 27 patients who had undergone anterior cruciate ligament reconstruction with the middle third of the homolateral quadriceps tendon. The examinations were performed on two MRI units: a permanent 0.2-Tesla dedicated magnet (Artoscan, Esaote Italy) and whole-body 1.5-Tesla superconducting magnet (Signa, GE Medical Systems Milwaukee, Winsconsin USA). Axial, sagittal and coronal images were acquired with SE, GE and STIR fat suppression sequences. The examinations were performed 1, 3, and 6 months post-operatively in 16 patients, and 1 and 3 post-operatively in 11 patients. The same arthroscopic surgical technique was employed in all patients, with 20 cases of tibial mono-tunnel femoral semi-tunnel, and 7 cases with tibial bi-tunnel technique. All patients were assessed by arthrometric and clinical tests after surgery. Bioabsorbable interference screws were used for tibial fixation in all patients and metallic interference screws were used for femoral graft fixation in 8 patients. RESULTS: In all cases MRI correctly visualised the tunnel positions, the articular portion and the bone-portion of the graft inside the tibial and femoral tunnels. The absence of paramagnetic artefacts in the tibia allowed complete visualisation on the axial, sagittal and coronal MRI images with optimal spatial and contrast resolutions. In 6 cases, the presence of metal residues from the surgical cutter prevented correct evaluation of femoral tunnel content. No new graft or articular lesions were found. In 18/27 cases peri-focal marrow edema around the tibial tunnel had disappeared 3 months after surgery. The process of synovial incorporation was judged to be correct in all cases. DISCUSSION: The use of anterior cruciate ligament reconstruction with the quadriceps tendon is a important innovation given the size of the harvested material and the possibility of completely filling the osseous tunnels, without interposition of synovial proliferation or fluid collection between tendon and bone, as confirmed by MRI. Furthermore, the use of non-metallic screws allows MRI evaluation of tunnel content and oedema in the spongy bone around the tunnel. The study of the double tibial tunnel requires specific obliqueness in the coronal plane scans. CONCLUSIONS: This arthroscopic technique for anterior cruciate ligament reconstruction allows thorough MRI evaluation of all portions of the transplant, and in particular those coursing within the tibia and femur. The absence of bone oedema around the tunnels and synovial proliferation within the tunnels may be predictive of faster healing and complete bone incorporation of the grafts.

Adult↗
PubMed2001

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.

Allosteric Regulation↗
PubMed2001

Fast coordination changes in cytochrome c do not necessarily imply folding.

Folding of globular proteins occurs with rates that range from microseconds to minutes; consequently, it has been necessary to develop new strategies to follow the faster processes that exceed stopped-flow capabilities. Rapid photochemical methods have been employed to study the rate of folding of reduced cytochrome c. In this protein, the iron of the covalently bound heme binds a His and a Met, proximal and distal. Unfolding by guanidine or urea weakens the Fe-Met bond, and the reduced unfolded cytochrome c easily binds CO and other heme ligands, which would react slowly or not at all with the native protein. Therefore in the presence of CO, reduced cytochrome c unfolds at lower denaturant concentrations than in the absence of this ligand, and rapid photochemical removal of CO from unfolded cytochrome c, is expected to trigger at least an incomplete refolding. This approach is complicated by the breakage of the proximal His-Fe bond that may occur as a consequence of CO photodissociation in the unfolded cytochrome c because of the so-called base elimination mechanism. Rebinding of CO to the four-coordinate heme yields kinetic intermediates unrelated to folding. Our hypothesis is supported by parallel observations carried out with protoheme and microperoxidase.

Animals↗
PubMed2001

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

Carbon Monoxide↗
PubMed2001

The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties.

Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa.

Binding Sites↗
PubMed2001

Photochemically induced electron transfer.

Biochemical reactions involving electron transfer between substrates or enzyme cofactors are both common and physiologically important; they have been studied by means of a variety of techniques. In this paper we review the application of photochemical methods to the study of intramolecular electron transfer in hemoproteins, thus selecting a small, well-defined sector of this otherwise enormous field. Photoexcitation of the heme populates short-lived excited states which decay by thermal conversion and do not usually transfer electrons, even when a suitable electron acceptor is readily available, e.g., in the form of a second oxidized heme group in the same protein; because of this, the experimental setup demands some manipulation of the hemoprotein. In this paper we review three approaches that have been studied in detail: (i) the covalent conjugation to the protein moiety of an organic ruthenium complex, which serves as the photoexcitable electron donor (in this case the heme acts as the electron acceptor); (ii) the replacement of the heme group with a phosphorescent metal-substituted porphyrin, which on photoexcitation populates long-lived excited states, capable of acting as electron donors (clearly the protein must contain some other cofactor acting as the electron acceptor, most often a second heme group in the oxidized state); (iii) the combination of the reduced heme with CO (the photochemical breakdown of the iron-CO bond yields transiently the ground-state reduced heme which is able to transfer one electron (or a fraction of it) to an oxidized electron acceptor in the protein; this method uses a "mixed-valence hybrid" state of the redox active hemoprotein and has the great advantage of populating on photoexcitation an electron donor at physiological redox potential).

Biophysics↗
PubMed2001

Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions.

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.

Aldehydes↗
PubMed2001

[Osteo-odonto-kerato-prosthesis. Radiographic, CT and MR features].

PURPOSE: Osteo-odonto-keratoprosthesis, a complex eye surgery technique devised by Strampelli, provides a valuable opportunity to restore vision in patients with severe corneal opacification (chemical or thermal burns, bullous keratopathy, severe keratitis, consequences of perforating injuries) in whom corneal transplant or the insertion of synthetic prostheses is contraindicated because of the high risk of rejection. Successful implantation of corneal prostheses in these patients was clearly dependent on the use of perfectly biocompatible materials to support the optic. Strampelli demonstrated that thin autologous tooth sections, complete with alveolar-dental ligament fulfilled these requirements, and integrated perfectly with the eye tissues without any risk of rejection. This study aims to present the radiological aspects and postoperative outcome of 13 patients who received osteo-odonto-keratoprosthesis (bilateral in 11 cases and monolateral in 2) evaluated by plain radiography, CT and MRI. MATERIAL AND METHODS: Between 1993 and 2001 we evaluated 13 patients who had undergone Strampelli's osteo-odonto-keratoprosthesis, using CT, plain radiography and MRI. All patients were examined by plain radiography; 11 patients were also examined by CT and 8 also by MRI. The time interval between surgery and the radiological evaluation ranged from 3 to 13 years with a mean follow-up of 5 years and 9 months. RESULTS: All patients underwent periodic clinical and imaging examinations in the post-operative period to evaluate the osteo-dental implant and to study trophism of the transplant. No post-operative complications, either cicatricial, inflammatory or of any other nature, were clinically suspected. Only two patients showed partial reabsorption of the osteo-dental lamina - evident both on plain film and CT - 10 and 12 years after surgery. Vision was restored in all the patients, with visual acuity of 10/10 in 7 cases. DISCUSSION AND CONCLUSIONS: Plain radiography allows to correctly evaluate the position of the prosthesis and detect possible displacements or variations in thickness, but it fails to visualize intraocular soft tissues. Besides allowing visualization and study of the prosthesis, CT also allows optimal evaluation of the intra-orbital structures and early detection of the presence and extension of inflammatory complications that may undermine outcome of the procedure. MRI is similar to CT in its capacity to evaluate intra-orbital tissues, but has the advantage of allowing greater contrast resolution thanks to the use of different types of sequences. However, because of the long image acquisition times, this method is subject to movement artifacts that are less evident in the CT examination which, especially if performed using the spiral technique, has very short image acquisition times.

Biocompatible Materials↗
PubMed2001

[Spontaneous remission of osteochondritis dissecans in 8 pediatric patients undergoing conservative treatment].

PURPOSE: Ostheocondritis dissecans lesions of the knee has a poor prognosis if certain signs are present at MRI. Clinical course is dependent on the site and extension of the lesions, and the age of the patient. In children, certain MRI findings similar to those found in the adult have a different prognostic significance, and in some case the condition can be cured by conservative treatment. By means of MR follow-up examinations performed at 6, 12 months, and in 2 cases, 24 months, we evaluated up the evolution of 8 osteochondritis dissecans treated conservatively with rest and antinflammatory drugs. MATERIAL AND METHODS: 7 pediatric patients (mean age 13 years) with 8 lesions of osteochondritis dissecans (bilateral in one cases) underwent MR follow-up examinations. In all cases the maximum longitudinal diameter of the lesion focus did not exceed 2 cm and at the first MR control the cartilage was intact. The MR examinations were performed using a dedicated low field magnet and a permanent low field magnet. RESULTS: MR allowed us to correctly identify both the origin and the extent of the lesion in all the patients. The follow-up examinations performed after 6, 12 and 24 months, showed complete disappearance of the osteochondritis dissecans focus in 6 cases whereas two lesions showed a 50% reduction of their extent. DISCUSSION AND CONCLUSIONS: Magnetic Resonance currently represents the gold standard for the evaluation of osteochondritis dissecans focus because, unlike other imaging techniques which are not able to identify the lesion it enables to establish an early diagnosis. Although the small number of the lesions examined only allows preliminary conclusions to be drawn, we have shown that in pediatric patients conservatively treated osteochondral lesions may evolve favourably. However, in all the cases examined the cartilage covering the lesion was intact. Favourable prognostic factors at MR are: stability of the transchondrial fragment and infraspongious edema at the adjacent bone. Peripheral hypervascularization may represent an attempt and spontaneous heading, as occurred in our cases.

Adolescent↗
PubMed2001

[New femoral fixation system for tendon transplantation in ACL reconstruction. Preliminary experience with MR imaging].

INTRODUCTION: The Authors describe the MR aspects of a new femoral fixation technique which has been recently introduced in anterior cruciate ligament recontructive surgery. This fixation device is composed of bioabsorbable material and of a thin thread which measures about 2,8 mm in diameter and 3,5 cm in length. This thread is positioned via the lateral transcondylar approach using the same arthroscopic technique as is used to position the transcondylar interference screw. By means of this technique it is possible to fix all the types of autologous grafts which are presently employed: patellar tendon, flexor tendons and quadriceps tendon. In MR, the absence of paramagnetic artifacts allows to detect also the graft portion inside the femoral bone tunnel and to acquire very accurate images. MATERIAL AND METHODS: By means of MR examination, we have studied 16 patients of either sex (11 M/ 5 F) with an age ranging from 18 to 37 years (mean age: 28,9 years). All of them underwent an anterior cruciate pro-ligament reconstruction by employing autologous tendons: the patellar tendom (=9), the quadriceps tendon (=4) and the flexor tendons (=3). By means of dedicated MR equipment at low-field intensity and with permanent magnet (Artoscan 0,2 T, Esaote, Genoa, Italy), every patient underwent follow-up controls during a period of time ranging from 1 to 9 months after the operation. We employed T1-weighted and T2-weighted spin-echo, gradient-echo and STIR sequences with fat tissue suppression. Besides the standard axial, sagittal and coronal planes, we employed a coronal plane with doubt obliquity which runs both along the axial plane and along the sagittal one with the aim of visualizing the whole graft on a single scanning plane including completely either the intra-articular portion or the bone tunnel portion. RESULTS: In none of these cases we did detect para-magnetic artifacts which could prevent us from performing a complete assessment either of the fixation device or of the content of the femoral tunnel. The tendinous graft was studied along the hole intra-articular length and inside the portions of the femoral and tibial bone tunnel. For this purpose the images acquired on oblique plane along the roof of the intercondylar notch were very useful. No patient reported significant complications, apart from two cases of maniscus everlasting pain as regards medial fibrocartilaginous sutures and one case of residual pain which concerns the anterior region of the patellar tendinous donor site. The evaluation form of the International knee Document Committee shows that in clinical terms all the patients achieved good results (=7) or excellent results (=9). Overall, we detected no new grant rupture or any functional limitation of the flexor-extension. DISCUSSION AND CONCLUSIONS: The advent of small-size bioabsorbable synthetic materials that are compatible with the MR equipment at out disposal, make it easier to obtain a good visualization of all the portions of the anterior cruciate tendinous ligament graft. By employing suitable sequences as well as scanning planes, MR is the only technique which is able to meet any operated patient's requirements and in particular those one which concern the development state of his/her recovery progress as well as the grant-taking. Follow-up studies will be necessary, however, to determine the real evolution as well as the complete integration of these reconstructions.

Adolescent↗
PubMed2000

Molecular mode of interaction of plant amine oxidase with the mechanism-based inhibitor 2-butyne-1,4-diamine.

2-Butyne-1,4-diamine (DABI) is a mechanism-based inhibitor of copper-containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is approximately 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p-dimethylaminobenzaldehyde and p-dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p-dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped-flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4-amino-2-butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.

Amine Oxidase (Copper-Containing)↗
PubMed2000

The oxidation and reduction reactions of bovine serum amine oxidase. A kinetic study.

The presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted.

Amine Oxidase (Copper-Containing)↗
PubMed2000

[Spiral CT and MR in injuries of the ankle and the foot].

Conventional radiography is the first radiological examination in injuries of ankle and foot, but often it has normal findings or it is difficult to execute for critical conditions of patients. Our purpose was to assess the value of CT and MRI in injuries of ankle and foot; we retrospectively reviewed the MR and CT images from 48 patients (age range 18-55 years), whose 16 had a major trauma with other skeletal segment lesions and the remaining a minor trauma limited to ankle and foot. On the basis of our results and experience, we think that in major trauma CT and MRI have similar findings about preoperative evaluation of fractures even if MR better identifies ischemia of fragments, subcortical contusions and lesions of ligaments, capsula or tendons. In minor trauma, MR is superior to CT because it can diagnose lesions of bone and periarticular structures that are difficult or impossible to identify with other instrumental examinations.

Adolescent↗
PubMed1999

Modulation of ligand binding in engineered human hemoglobin distal pocket.

Functional and structural studies on hemoglobin and myoglobin from different animals and engineered variants have enlightened the great importance of the physico-chemical properties of the side-chains at topological position B10 and E7. These residues proved to be crucial to the discrimination and stabilisation of gaseous ligands. In view of the data obtained on the high oxygen affinity hemoglobin from Ascaris worms and a new mutant of sperm whale myoglobin, we selected the two mutations Leu B10-->Tyr and His E7-->Gln as potentially relevant to control ligand binding parameters in the alpha and beta-chains of human hemoglobin. Here, we present an investigation of three new mutants: HbalphaYQ (alpha2YQbeta2A), HbbetaYQ (alpha2Abeta2YQ) and HbalphabetaYQ (alpha2YQbeta2YQ). They are characterised by a very low reactivity for NO, O2 and CO, and a reduced cooperativity. Their functional properties are not inconsistent with the behaviour expected for a two-state allosteric model. Proteins with these substitutions may be considered as candidates for the synthesis of a possible "blood substitute", which should yield an O2 adduct stable to autoxidation and slowly reacting with NO. The mutant HbalphabetaYQ is particularly interesting because the rate of reaction of NO with the oxy and deoxy derivatives is reduced. A structural interpretation of our data is presented based on the 3D structure of deoxy HbalphabetaYQ determined by crystallography at 1.8 A resolution.

Allosteric Regulation↗
PubMed1999

CuI-semiquinone radical species in plant copper-amine oxidases.

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

Amine Oxidase (Copper-Containing)↗