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A Bellelli

Publications and source records attributed to A Bellelli.

At least 37 records · Page 2Linked to original sources
PubMed1999

Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.

Alanine↗
PubMed1999

Magnetic resonance imaging of tunnel placement in posterior cruciate ligament reconstruction.

The aim of this study was to define a reproducible method for evaluating posterior cruciate ligament (PCL) reconstructions using magnetic resonance imaging (MRI). A 2-fold investigation was performed. In part I, the "footprints" of an intact PCL were located on MRI and their coordinates were defined. Measurements were made on the images of 50 subjects using axial, coronal, and sagittal planes. Interobserver variability was calculated by averaging the measurements of the 2 reviewers and using the Kappa coefficient. Three points of reference were located: tibial attachment on the tibial axial plane, and two femoral attachments on the sagittal and coronal oblique planes. In part II, stability of 20 PCL reconstructions with a bone-patellar tendon-bone (BPTB) autograft were evaluated and scored using the IKDC evaluation form after a 2-year follow-up. Stability was evaluated clinically and instrumentally using a KT-2000 arthrometer at 89 N with the knee flexed at a neutral quadriceps knee angle of approximately 70 degrees . Seven cases were graded A (0 to 2 mm), 11 graded B (3 to 5 mm), and 2 graded C (6 to 10 mm). All patients had an MRI after an average of 16 months (range, 12 to 24 months, 2 SD). The previous measurements from part I of the study were used to make a correlation between achieved stability and tunnel location. A 1-factor analysis of variance (ANOVA), nonparametric ANOVA, and the Fisher Exact test were used to determine if clinical outcome of the 3 groups was influenced by graft placement. At MRI evaluation, excessive deep placement was observed in 4 cases and a correlation between improper femoral tunnel location and stability was statistically significant (P < .05). A correct placement of tibial tunnel was observed in all patients. In our analysis, proper location of the femoral tunnel seems to be more critical and difficult to achieve than tibial tunnel placement, probably because of the lack of specific anatomic landmarks during surgery.

Adolescent↗
PubMed1999

Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy.

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O2 may account for the puzzling (20-fold) increase of the KM for O2 reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.

Fluorescence↗
PubMed1999

[Arthroscopic reconstruction of the posterior cruciate ligament. Magnetic resonance study for bone tunnel positioning and comparison with clinical results].

INTRODUCTION: The success of arthroscopic cruciate ligament reconstruction depends on several factors, such as patient selection, correct surgical execution, and postoperative rehabilitation. Technical considerations include graft choice, positioning, fixation, intercondylar notch enlargement, and new ligament tensioning. Graft acceptance is effected by all these factors. Tunnel position is of great importance both for biomechanical reasons and optimal function of the new ligament, and to avoid stress, friction, abnormal strain, and/or damage to the reconstructed ligament. Many orthopedic and radiographic literature studies discuss the exact site of anterior cruciate ligament insertion for the best possible anatomical reconstruction. In contrast, the debate over insertional area and anatomical landmarks is open for the posterior cruciate ligament (PCL), because of the difficult execution of this type of reconstruction and the smaller number of candidates. MATERIAL AND METHODS: Fifty patients with a healthy PCL underwent MRI of the knee for other diagnostic reasons and we measured the position of PCL insertion at the tibial and femoral condyles. We also examined with MRI 20 surgical patients with a reconstructed PCL. Graft position was assessed with the same method and the results compared with physical findings of joint stability and the IKDC form score. RESULTS: Three main landmarks were found on standard axial, coronal and sagittal MR images: T1 on the tibia, and F1 and F2 on the femur. These points refer to the fibrous ligament center and designate the medial, middle and lateral portion of the tibial plateau, as well as the anterior/posterior and high/low positions on the roof of the intercondylar notch and anteromedial side of the medial condyle, respectively. According to these data, the midline position, whether slightly medial or lateral, of tibial insertion, was clinically less important. On the contrary, correct femoral tunnel positioning was found to effect subsequent joint stability and prompt rehabilitation. CONCLUSIONS: This method for MR measurement is easy and repeatable, and can be used for surgical planning and patient follow-up. We found it extremely useful for the correct positioning of bone tunnels, particularly the femoral condyle, in all cases.

Arthroscopy↗
PubMed1999

[Synovial healing in reconstructed cruciate ligaments. Our personal experience compared in single interventions and combined reconstructions].

INTRODUCTION: The healing process of tendon grafts used in cruciate ligament reconstruction is called ligamentization. The tendon structural architecture changes progressively into the histological appearance of normal ligament. Amiel and Clancy have demonstrated that this process is time-dependent in anterior cruciate ligament (ACL) reconstruction, the tendon graft taking one year to become similar to a normal cruciate ligament. Three different maturation phases of anterior cruciate graft ligamentization can be seen at MRI: periligamentous proliferation, intraligamentous proliferation and definitive healing. We report our MR findings in the reconstruction of single ACL, posterior cruciate ligament (PCL), and of both ACL and PCL. MATERIAL AND METHODS: January 1995 to January 1999 we selected 60 patients submitted to arthroscopic cruciate ligament reconstruction. The ACL was reconstructed with the patellar tendon in 22 cases and the PCL in 23; fifteen patients underwent double reconstruction of ACL, with hamstring tendons, and PCL, with patellar tendons. Fifty-five patients were followed-up with MRI: 45 with a dedicated permanent magnet (Artroscan, Esaote Biomedica, Genoa, Italy) and 10 with a permanent low-field unit (AIRIS, Hitachi, Japan); the same technical parameters were used in all cases. Forty of 55 patients were examined at 1, 3, 6, 9 and 12 postoperative months, while 15 were followed-up longer (12 to 36 months postoperatively). At the first follow-up MR examination, 5 patients (2 ACL and 3 PCL) were excluded for incorrect tunnel position (1 case), hypertrophic scarring (2 cases) and new trauma (2 cases). MR findings were compared with clinical data of joint stability. RESULTS: MRI showed the different stages of the healing process in 20 ACL patients: proliferating soft tissue around the graft with the low signal intensity typical of tendons (stage I, 1-3 months postoperatively), the graft becomes progressively hyperintense (stage II, 3-9 months postoperatively), and finally the low signal intensity indicating completed ligamentization (stage III, 12 months postoperatively). As for PCL reconstructions, MR findings were similar but the process took longer, even 24 months. Finally, in the 15 cases of double reconstruction, both grafts were difficult to distinguish on T1-weighted images for a very long time (24-36 months postoperatively). DISCUSSION AND CONCLUSIONS: Relative to the ACL, ligamentization takes longer for PCL and combined ACL and PCL reconstructions, probably because the morphological changes in PCL and double grafts may be impaired by many factors, such as gravity, long bone tunnels, hemarthrosis, hyperplastic synovial reaction in the intercondylar notch; also, rehabilitation protocols differ in ACL from PCL patients. No signs of instability were found at physical or arthrometric examinations. MRI demonstrates the different stages of ligamentization and thus proves a useful tool for postoperative follow-up in cruciate ligament reconstructions. Contrast-enhanced (Gd) studies are reserved to the cases of graft impingement or if other abnormalities are suspected.

Anterior Cruciate Ligament↗
PubMed1998

Fast-reacting thiols in rat hemoglobins can intercept damaging species in erythrocytes more efficiently than glutathione.

The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mM), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t1/2 <100 ms; slow reacting (sHbSH), with t1/2 30-50 s; and very slow reacting (vsHbSH), with t1/2 180-270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125beta(H3)) conjugates with DTNB 100 times faster than glutathione and approximately 4000 times more rapidly than (v)sHbSH (Cys-13alpha(A11) and Cys-93beta(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (approximately 6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123beta(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mM, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.

Animals↗
PubMed1998

Intermediates in the catalytic cycle of lentil (Lens esculenta) seedling copper-containing amine oxidase.

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Stabilization of the T-state of ferrous human adult and fetal hemoglobin by Ln(III) complexes: a thermodynamic study.

The effect of the lanthanide(III) complexes [Gd(1,4,7,10-tetraazacyclododecane-N,N',N", N"'-tetrakis(methylenephosphonate))]5- (Gd-DOTP) and La-DOTP on the oxygen binding and spectroscopic properties of human adult and fetal hemoglobin (HbA and HbF, respectively) has been investigated. The affinity of Gd-DOTP and La-DOTP for oxygenated HbA (HbAO2; KHbAO2 = 2.6 x 10(-3) M) is closely similar to that observed for Ln(III) complexes association to nitrosylated HbA (HbANO KHbANO = 1.8 x 10(-3) M) and to aquo-met HbA (met-HbA; Kmet-HbA = 1.9 x 10(-3) M), being lower than that determined for Gd-DOTP and La-DOTP binding to the deoxygenated form of the tetramer (HbAd; KHbAd = 3.0 x 10(-4) M). The affinity of Gd-DOTP for deoxygenated HbF (HbFd; KHbFd = 9.5 x 10(-4) M) and oxygenated HbF (HbFO2; KHbFO2 = 3.7 x 10(-3) M) is lower than that observed for Ln(III) complexes association to HbAd and HbAO2, respectively. Gd-DOTP and La-DOTP bind to HbA and HbF with a 1:1 stoichiometry per tetramer. Increasing Gd-DOTP and La-DOTP concentration, oxygen affinity for HbA decreases (i.e. P50 increases), this effect being minor for HbF. Upon binding of Ln(III) complexes to HbANO, the X-band EPR spectrum and the absorption spectrum in the Soret region display the characteristics which have been attributed to the T-state of the ligated tetramer. These results represent a clear cut evidence for the specific binding of Gd-DOTP and La-DOTP to the 2,3-D-glycerate bisphosphate (BPG) pocket (i.e. at the dyad axis, in between the beta-chains) of HbA and HbF. The effect of Ln(III) complexes on the ligand binding and spectroscopic properties of HbA and HbF is reminiscent that of BPG, the physiological modulator of human Hb action.

Adult↗
PubMed1998

[Cicatrization of complete traumatic lesions of the posterior cruciate ligament. Magnetic resonance follow-up of 10 cases and a proposal for modification of Gross classification].

INTRODUCTION: Traumatic injuries of the knee ligaments present in various degrees of severity from interstitial sprains to complete ligament tears. The femoral insertion of the anterior cruciate ligament (ACL) is susceptible to post-traumatic ischemic changes secondary to interrupted vascular supply from the tibial insertion. In comparison, the posterior cruciate ligament (PCL) has more extensive vascularization from the posterior geniculate arteries, which allows better healing of these injuries with fibrous scar tissue. MRI of a scarred PCL injury shows lower than normal signal intensity, which post-traumatic changes in signal intensity are well described in the literature. In my personal experience with more than 5500 MR examinations of the knee, PCL traumatic injuries secondary to motor vehicle accidents or sports injuries are a frequent finding. Many of these patients are completely asymptomatic for articular instability. MATERIAL AND METHODS: From November, 1994, to July, 1997, a direct MR follow-up was proposed to patients with PCL traumatic tears. Only the patients examined in our hospital were included in this study because the MR scans were performed using the same unit, matrix, FOV, slice thickness and sequences. Twenty patients had acute traumatic injuries of the PCL. Four were not examined because of an additional traumatic episode and four were not included because they were operated on. Ten patients were thus enrolled: three women and seven men whose age ranged 23 to 41 years (mean: 35.7). MR follow-up ranged 9 to 36 months (average: 14.2). RESULTS: At the first MR examination, PCL tears were classified as follows: Gross class II (1 patient) and Gross class III (9 patients). Follow-up MRI showed no PCL in 2 patients (similar to ischemic degeneration in complete ACL tears), focal interligament changes within a continuous PCL in 2 patients and normal PCL in 6 patients. The trauma was caused by a motor vehicle accident with the knee flexed in 8 patients and during hyperflexion in sports activity (skiing or soccer) in 2 patients. Five patients presented with knee instability and subjective complaints, such as pain and difficulty in climbing stairs, walking, running, or performing sports activity. Follow-up MRI showed natural repair of the PCL traumatic injury in 5 patients, with absolutely no knee instability. CONCLUSION: Even though the number of patients in this study is limited, follow-up MRI confirms the possibility of scar tissue repair in PCL post-traumatic injuries, supporting primary conservative treatment.

Accidents, Traffic↗
PubMed1997

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

4-Chloromercuribenzenesulfonate↗
PubMed1997

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis.

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d1 heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd1 NiR in which Tyr10 has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fulop, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369-377) that the topologically homologous Tyr25 plays a crucial role in controlling the activity of the cd1 NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr10 with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe3+ of the d1 heme is provided by different side-chains in different species.

Amino Acid Sequence↗
PubMed1997

The unusual stability of saporin, a candidate for the synthesis of immunotoxins.

Saporin, a monomeric protein extracted from the seeds of Saponaria officinalis, is an enzyme capable of specific depurination of the eukaryotic ribosomes. Because of its toxicity, saporin proved useful for the synthesis of immunotoxins, chimeric conjugates of a toxin and an antibody specifically directed against cancer cells or other targets. In this paper we report a study of the structural properties of saporin in the presence of denaturing agents and/or proteolytic enzymes. We found that saporin is extremely resistant to denaturation by urea or guanidine (up to 4 M), even at relatively high temperature (up to 55 degrees C). Moreover a structural change detected as a reduction of the fluorescence emission of the single Trp residue is reversible and is not paralleled by changes of the far UV CD spectrum, suggesting that even under harsh experimental conditions unfolding is limited. In good agreement with these results, guanidine-treated saporin is not attacked by proteolytic enzymes. The remarkable resistance of saporin to denaturation and proteolysis suggests this protein as an ideal candidate for biotechnological applications.

Circular Dichroism↗
PubMed1997

Arthroscopic absence of the long head of the biceps tendon.

Absence of the long head of the biceps tendon was detected during arthroscopic evaluation of shoulder pain in a weight-lifter. Magnetic resonance imaging and ultrasound showed the presence of a hypoplastic tendon with altered depth and width of the bicipital groove. The tendon-glenoid insertion shown by magnetic resonance imaging was apparently normal.

Adult↗
PubMed1997

[Dynamic magnetic resonance of the knee. Considerations on techniques and anatomy with a magnetic resonance system with open magnet].

MRI is currently considered the best technique to study the joints--knee joints in particular. Most studies are performed with the knee straightened out or slightly flexed, which position is mandatory with contemporary MR units. Recently, however, the MR equipment for limb studies and the larger tunnels of total body magnets have permitted partially dynamic knee studies, up to 30-40 degrees flexion; this joint excursion cannot be exceeded. In 1996, Muhle and Niitsu demonstrated that dynamic MRI can be useful to study cruciate ligament injuries, some types of meniscal injuries, all femoropatellar conditions and, mostly, cruciate ligament reconstruction. We used an open magnet MR unit for thorough functional dynamic knee studies, acquiring the images at 0-120 degrees flexion. The open magnet MR unit permits free joint movements. We examined 25 subjects: 10 healthy volunteers and 15 patients with meniscal, capsuloligamentous, femoropatellar or synovial conditions. This technique schedules axial, coronal or sagittal images, according to the diagnostic suspicion, suitably directed with the patient in lateral decubitus and with the knee flexed to maximum joint excursion. Dynamic MR findings are easily transferred to an X-ray film, so that the radiologist can send them to the orthopedist with no need of any videotape or TV take. The current cine MR programs display the dynamic images of the moving joint on an MR monitor, but this option is not yet widely available. The quality of dynamic MR studies is exactly the same as that of conventional images, with a particular emphasis on the small diagnostic details of the injuries. We report our preliminary experience with this technique, indicating some possible clinical applications of dynamic MRI which permits thorough studies of knee flexion-extension.

Humans↗
PubMed1996

Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa.

1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7 x 10(5) s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c-->d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.

Electron Transport↗