Erythrocyte's aging in microgravity highlights how environmental stimuli shape metabolism and morphology.

The determination of the function of cells in zero-gravity conditions is a subject of interest in many different research fields. Due to their metabolic unicity, the characterization of the behaviour of erythrocytes maintained in prolonged microgravity conditions is of particular importance. Here, we used a 3D-clinostat to assess the microgravity-induced modifications of the structure and function of these cells, by investigating how they translate these peculiar mechanical stimuli into modifications, with potential clinical interest, of the biochemical pathways and the aging processes. We compared the erythrocyte's structural parameters and selected metabolic indicators that are characteristic of the aging in microgravity and standard static incubation conditions. The results suggest that, at first, human erythrocytes react to external stimuli by adapting their metabolic patterns and the rate of consumption of the cell resources. On longer timeframes, the cells translate even small differences in the environment mechanical solicitations into structural and morphologic features, leading to distinctive morphological patterns of aging.

Switching between the alternative structures and functions of a 2-Cys peroxiredoxin, by site-directed mutagenesis.

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.

Neurocognitive rehabilitative approach effectiveness after anterior cruciate ligament reconstruction with patellar tendon. A randomized controlled trial.

BACKGROUND: A proper knee rehabilitation after a surgical reconstruction of the anterior cruciate ligament (ACL) should start immediately after the injury and it should be focused on recovery of symmetry, proprioception, swelling reduction, gait training, hyperextension exercises, and even mental preparation. AIM: Aim of this study was to test a neurocognitive rehabilitative approach based on proprioceptive exercises and proper motor strategy choices, compared with conventional rehabilitation, assessing baropodometric, gait and clinical changes. DESIGN: Randomized controlled trial. SETTING: Ambulatory University Centre. POPULATION: Fourteen subjects (27.9±5.2 years) underwent to a surgical reconstruction of ACL were divided into the two groups. METHODS: The subjects were randomly assigned into a group who received a specific neurocognitive and perceptive rehabilitation treatment (TG), and into a control group who received the common physical therapy (CG). The following outcome measures were assessed pre-intervention, one, three and six months later: static and dynamic baropodometry, Visual Analog Scale for pain, Short Form SF-36, Range of Motion, trophism of thigh region, edema, Manual Muscle Test, magneto-resonance imaging assessment. RESULTS: Lower impairment was observed in TG in respect of CG in terms of load asymmetry during static baropodometry (from 7% to 3% vs. from 10% to 7%, interaction time per treatment: P=0.037), less wide steps during gait (effect size=1.05 vs. 0.38 for CG), swelling (treatment effect: P=0.012). A significantly higher improvement (from 35% to 100%) in terms of SF-36 was recorded only in TG for physical activity (P=0.027). CG showed a quite higher walking speed (treatment effect: P=0.049). CONCLUSION: Even if further studies are needed on larger samples, the obtained results showed that a neurocognitive rehabilitative approach could be an effective treatment after ACL-reconstruction: in TG we observed a more rapid load symmetrization, the reduction of step width and a more rapid resolution of edema. CLINICAL REHABILITATION IMPACT: Posture, gait, clinical features and quality of life could benefit from a neurocognitive rehabilitation after ACL surgical reconstruction.

Pulmonary ultrasonography in the follow-up of respiratory distress syndrome on preterm newborns. Reduction of X-ray exposure.

PURPOSE: Respiratory distress syndrome (RDS) is the most common cause of respiratory failure in preterm neonates, whose lungs are often immature. The diagnosis and follow-up are based on clinical and radiographic findings. Due to the problem of air artifacts, ultrasonography (US) is not used routinely in the diagnosis of lung diseases. However, when the alveolar air content decreases, as it does in RDS, characteristic patterns appear that can be observed during US lung examinations. The aim of this study was to determine whether the use of chest radiographs in neonates with RDS could be reduced by the routine use of chest US for follow-up examinations. MATERIALS AND METHODS: From April through September 2008, were enrolled all preterm newborns, with very low birth weight (VLBW), consecutive admitted in NICU with clinically and radiologically diagnosed RDS. We performed lung ultrasound examination in this patients. Video-taped US examinations were done every 8-12 h until clinical resolution of the disease was observed. Chest X-rays were performed only in unclear cases. We compared the number of chest radiographs obtained in the NICU during this period and during the preceding six months. RESULTS: 105 serial US lung examinations were performed in 21 preterm infant with clinically and radiologically diagnosed RDS. US lung examinations revealed "comet-tail" artifacts that were compact, diffuse, and symmetrically distributed throughout both lung fields. In 8 cases, the pleural line was also extensively thickened and irregular, and in 7 cases multiple subpleural hypoechoic areas indicative of lung consolidation were observed (mainly on posterior and lateral scans). The mean number of chest radiographs per infant performed in the NICU during the study period was significantly lower than that of the preceding six months (2.6 ± 1 versus 3.8 ± 1.5; p < 0.05). CONCLUSIONS: Chest ultrasound is a valid alternative for the follow-up of VLBW infants with RDS, which can decrease the need for chest X-rays and reduce patient exposure to ionizing radiation.

Cl- and F- anions regulate the architecture of protofibrils in fibrin gel.

Ischemic heart disease is the leading cause of serious morbidity and mortality in Western society. One of the therapeutic approaches is based on the use of thrombolitic drugs that promote clot lysis. Even if the mechanisms leading to clot lysis are not completely understood, it is widely accepted that they depend on the complex biochemical reactions that occur among fibrin fibers and fibrinolitic agents, and by their ready diffusion into the fibers. Here we investigate the effects of specific anions on the architecture of protofibrils within fibrin fibers in fibrin gels prepared in a para-physiological solution. The results obtained through small-angle X-ray scattering (SAXS) demonstrate that the characteristic axial and longitudinal repeat distances among protofibrils are strongly affected by the action of Cl(-) and F(-) anions.

Technical note: MRI device for active stress of the knee. The practical approach and preliminary data.

PURPOSE: The aim of this study was to test the potential application of a new nonmagnetic device capable of diagnostically evaluating stress of the knee joint during magnetic resonance imaging (MRI) investigation. MATERIALS AND METHODS: The Knee Loader prototype was applied to an MRI scanner with a 1.5-T superconducting magnet. Twenty healthy subjects were assessed in both baseline conditions and again after applying a known amount of force (in kg) to various joints. Standard MR techniques were employed. The degree of anterior and posterior tibial translation and variations in the width of the rim in varus and valgus were measured in millimetres. RESULTS: In 12 cases, the anterior drawer test demonstrated a rest-stress variation between 0 and 2 mm, and in two cases, the posterior drawer test was between 1 and 3 mm. In the four cases assessed at valgus angulations and the two cases assessed at varus angulations, the opening of medial and lateral compartment was between 1 and 2 mm. CONCLUSIONS: The preliminary data regarding this new MR method, performed using the Knee Loader prototype, demonstrate that it is possible to obtain functional images of the knee comparable with those obtained by traditional radiological techniques. Its potential advantage lies in the fact that, given the absence of ionising radiation, the investigation can be repeated as necessary, ensuring anatomical visualisation of all joint components under stress.

CO-MP4, a polyethylene glycol-conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats.

BACKGROUND AND PURPOSE: MP4 (Hemospan) is a Hb-based oxygen therapeutic agent, based on polyethylene-glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO-MP4 on myocardial infarct size following ischaemia and reperfusion in rats. EXPERIMENTAL APPROACH: Kinetic measurements of CO-MP4 binding were used to evaluate the effects of PEG modification on Hb subunit structure/function and to calculate CO-MP4 equilibrium constants. CO transport by CO-MP4 was shown by ligand (O2/CO) partitioning between MP4 and red blood cell (RBC)-Hb. Pharmacological effects of CO-MP4 were studied on myocardial infarction in rats. KEY RESULTS: CO binding kinetics show primary structural/functional effects on beta chains in MP4, with alpha chains maintaining the ability to undergo tertiary conformational transition. CO confers long-term, room-temperature stability and is able to rapidly re-equilibrate between MP4 and RBCs. In a rat model of myocardial infarct, in contrast to oxy-MP4, CO-MP4 reduced infarct size when administered prior to the induction of ischaemia. CONCLUSIONS AND IMPLICATIONS: MP4 PEGylation chemistry modifies the individual function of Hb subunits, but results in an overall CO equilibrium constant similar to that for unmodified Hb. CO-MP4 is able to deliver CO to the circulation and reduces ischaemia/reperfusion injury in rats, providing the first evidence for this drug as a CO therapeutic agent.

Swelling of the submandibular glands after administration of low-osmolarity contrast agent: Ultrasound findings.

Swelling of the salivary glands occurring after injection of iodine based contrast agent is a rare late adverse reaction. Only a few cases in the literature report such diagnostic findings. We present our color Doppler ultrasound findings in a case of swelling of both submandibular glands occurring after administration of iodinated contrast agent.

An unusual case of hematuria caused by a foreign body in the bladder: US and radiologic findings.

The presence of a foreign body in the bladder or urethra is generally the result of autoerotic acts, psychiatric problems, or therapeutic interventions. It can cause hematuria, pain, and abscess formation, which may lead the patient to seek medical care, but the diagnosis is not always easy. This report describes a man who presented with several months' history of hematuria. Ultrasonography revealed a foreign body in the bladder, and the diagnosis was confirmed by abdominal radiography. The object, which was endoscopically removed, proved to be the ceramic sheath of an endoscopic resector that had been used 2 years earlier for transurethral resection of the prostate.

The anti-schistosomal drug praziquantel is an adenosine antagonist.

The mechanism of action of praziquantel (PZQ), the drug of choice against schistosomiasis, is still unclear. Since exposure of schistosomes to the drug is associated with calcium influx and muscular contraction, calcium channels have been suggested as the target, although direct combination of PZQ with their subunits was never demonstrated. We report a hitherto unknown effect of PZQ, namely the inhibition of nucleoside uptake, as observed in living worms using radio-isotope labelled adenosine and uridine. This effect is clearly seen in schistosomes but is absent in mammalian cells in culture. Moreover it is a specific pharmacological effect seen exclusively with the active levo-R(-)stereo isomer of the drug, and is shared by at least one benzodiazepine having antischistosomal activity. This novel effect acquires significance given that schistosomes cannot synthesize purine nucleosides de novo. A possible relationship between this novel effect and the known action of PZQ on calcium channels is discussed, since adenosine is known to bind to specific receptors and to behave as an indirect antagonist of calcium release in mammalian cells. If calcium channels were correlated with adenosine receptors also in schistosomes, as they are in mammals, this would support the hypothesis that PZQ-induced calcium influx may be correlated to adenosine receptor blockade.

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.

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.

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.

[New femoral fixation system for tendon transplantation in ACL reconstruction. Preliminary experience with MR imaging]. / Nuovo sistema di fissazione femorale dei trapianti tendinei pro-LCA. Esperienza preliminare con RM.

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.

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.

[Osteo-odonto-kerato-prosthesis. Radiographic, CT and MR features]. / L'osteo-odonto-cherato-protesi. Aspetti radiografici, TC ed RM.

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.

[Spontaneous remission of osteochondritis dissecans in 8 pediatric patients undergoing conservative treatment]. / Risoluzione spontanea di 8 casi di osteocondrite dissecante in pazienti di età pediatrica trattati in maniera conservativa.

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.

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.

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.