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A Bellelli

Publications and source records attributed to A Bellelli.

At least 55 records · Page 3Linked to original sources
PubMed1996

Probing the alpha 1 beta 2 interface of human hemoglobin by mutagenesis. Role of the FG-C contact regions.

The allosteric transition of hemoglobin involves an extensive reorganization of the alpha 1 beta 2 interface, in which two contact regions have been identified. This paper concerns at the effect of two mutations located in the "switch" (alpha C3 Thr --> Trp) and the "flexible joint" (beta C3 Trp --> Thr). We have expressed and characterized one double and two single mutants: Hb alpha T38W/beta W37T, Hb beta W37T, and Hb alpha T38W, whose structure has been determined by crystallography. We present data on: (i) the interface structure in the contact regions, (ii) oxygen and CO binding kinetics and cooperativity, (iii) dissociation rates of deoxy tetramers and association rates of deoxy dimers, and (iv) the effect of NaI on deoxy tetramer dissociation rate constant. All the mutants are tetrameric and T-state in the deoxygenated derivative. Reassociation of deoxygenated dimers is not modified by interface mutations. DeoxyHb alpha T38W/beta W37T dissociate much faster. We propose a binding site for I- at the switch region. The single mutants binds O2 cooperatively; the double one is almost non-cooperative, a feature confirmed by CO binding. The functional data, analyzed with the two-state model, indicate that these mutations reduce the value of the allosteric constant LO.

Allosteric Site↗
PubMed1996

Kinetic and spectroscopic properties of the cyanide complexes of ferrous haemoglobins I and IV from trout blood.

The cyanide ion is a ligand of ferrous as well as ferric haemoproteins and this study presents a kinetic characterization of the dissociation of its complexes with the two main haemoglobin components from trout blood. Both these haemoglobins bind oxygen co-operatively at neutral or alkaline pH values but one of them is insensitive to pH and allosteric effectors (haemoglobin I, HbI) while the other (haemoglobin IV, HbIV) is strongly sensitive and shows the so-called Root effect (i.e. the incomplete oxygen saturation in air-equilibrated solutions at pH values of < 6.5). Comparison of the kinetics of dissociation of cyanide from ferrous forms of HbI and HbIV reveals that: (i) cyanide dissociates in both cases by a complex reaction, and, at least in the case of HbIV, this may be attributed to functional differences between the alpha and beta subunits; (ii) the reaction is only scarcely co-operative in HbI and not at all so in HbIV; and (iii) the Bohr and Root effects are not manifested in this reaction. The functional heterogeneity of ferrous alpha and beta chains of trout HbI has not been observed for any other ligand; moreover, the observation that co-operativity for cyanide dissociation is expressed by human haemoglobin but not by trout HbIV is surprising.

Animals↗
PubMed1996

A saporin-insulin conjugate: synthesis and biochemical characterization.

Saporin, a single-chain, non-cytotoxic, ribosome-inactivating protein from Saponaria officinalis, was chemically linked to the hormone insulin in a 1:1 complex. To follow by dynamic video microscopy the endocytosis and intracellular transport in vivo, a second covalent conjugate with a saporin derivative labelled with fluorescein isothiocyanate was also prepared. Both conjugates were characterized with reference to homogeneity, stoichiometry, optical spectroscopy and toxicity. Both were found to exhibit scarce toxicity toward both CHO and HEP G2 cells; optical video microscopy on living cells indicates that reduced toxicity may be (partly) due to a very limited binding of the saporin-insulin conjugate to membrane receptors. These results suggest a strategy for new possible covalent conjugates of saporin with alternative and specific macromolecular carriers.

Animals↗
PubMed1996

[Meniscal deformities associated with fractures of the tibial proximal extremity. Considerations in 7 cases].

Lateral tibial plateau fractures are a fairly frequent event in emergency clinical practice. In these fractures, when bone depression exceeds 5 mm, surgery is indicated. On the rule, conventional plain films combined with tomography can answer diagnostic questions about bone trauma. CT and MRI permit to study associated meniscocapsular injuries for better therapeutical management. Since February, 1991, we have examined 24 patients with tibial plateau fractures with conventional radiography and CT. CT was performed using thin sections, within 0 to 48 hours of the traumatic event. In our series, 7 patients had a lateral meniscal trauma associated with a fracture of the homologous tibial plateau; in all of these 7 women, surgery confirmed complete meniscal avulsion. In these cases, CT showed the following signs of meniscocapsular disinsertion: marked diastasis between capsular structure, popliteal tendon and meniscal profile; associated hypodense hemorrhagic fluid in the popliteal recess; inhomogeneous densitometry of the popliteal tendon resulting from hemorrhagic infarction. Furthermore, CT showed a characteristic and constant morphological alteration of the lateral meniscus with fibrocartilage deformation, that is with a wider or more narrow pattern relative to its normal "C"-like shape. We conclude that this morphological alteration of meniscal fibrocartilage, when associated with a tibial fracture, is a diagnostic CT sign of complete meniscal avulsion. This finding can be a useful integration to other CT signs of this meniscal injury, towards better and more complete therapeutical management.

Adolescent↗
PubMed1996

[Occult or unknown traumatic osteochondral lesions of the knee. Assessment of 19 cases studied with conventional radiology and magnetic resonance].

Occult fractures of the knee represent a major cause of posttraumatic knee pain in patients with negative plain films. These injuries can cause severe sequelae if they are not properly diagnosed and rapidly treated. In our series of 1330 exams of the knee we found 19 traumatic osteochondral injuries (1.5%). MR studies were performed with a dedicated permanent magnet (0.2 T) (Artoscan, Esaote Biomedica, Genoa, Italy). All the patients were submitted to conventional radiography (AP and LL projections). In 14 patients oblique plain films were also performed. The following osteochondral injuries were found: 2 purely chondral fractures, 5 occult fractures (4 tibial and 1 of the medial femoral condyle) and 12 plateau fractures of the tibial posterior margin (8 lateral and 4 medial). CT of the knee was performed in 3 patients only. In 19 patients MRI correctly showed the traumatic injury and depicted: fracture site, the morphologic characteristics of fracture line, cortical bone and articular cartilage involvement, the exact extent of marrow signal changes, the associated injury of menisci or cruciate ligaments. To conclude, MR sensitivity and diagnostic accuracy are excellent in the patients with traumatic injuries of the knee. In the patients with posttraumatic knee joint pain, MRI appears to be an extremely useful tool in the detection and assessment of bone and cartilage disorders.

Adolescent↗
PubMed1996

[Synovial cyst of the cruciate ligament. Findings with magnetic resonance in 8 symptomatic cases].

Intraarticular ganglion cysts are uncommon findings: only 30 cases have been reported since the first paper by Caan in 1924 and they were all associated with cruciate ligaments. Many different cystic or pseudocystic lesions are found in articular knee conditions: the most common cystic lesions are popliteal cysts (Baker's cysts), followed by synovial pseudocysts of the posterior cruciate ligament, meniscal cysts and, finally, ganglion cysts of the cruciate ligaments. In our series of 1600 knee MR exams carried out in our MR department since June, 1994, we have found 8 ganglion cysts of the cruciate ligaments. MR studies are always performed on a dedicated 0.2-T permanent magnet (Artoscan, Esaote Biomedica, Genoa, Italy). Five patients were operated on with arthroscopy. The ganglion cysts affected the anterior cruciate ligaments in 4 cases and the posterior cruciate ligaments in 4 cases. The symptoms were mainly pain radiating to the medial side and worsening in forced flexion or extension. The diagnostic suspicion was meniscal tears in 4 patients, chondral lesions in 3 and a loose intraarticular body in one patient. The shape and structure of ganglion cysts in the cruciate ligaments are clearly depicted with MRI. The ganglion cysts in the anterior cruciate ligaments are usually spindle-shaped and within the ligament, while those in the posterior cruciate ligaments have a well-defined outline, with multilocular appearance, and they are usually localized along the ligament, most often on the dorsal aspect. MR signal studies show intermediate signal intensity on SE T1-weighted images and markedly increased signal intensity on SE T2-weighted images. These typical patterns may change depending on lesion content, for instance in the presence of hemoglobin due to an associated angioma. The origin of ganglion cysts in the cruciate ligaments is still unknown, even though many theories have been suggested, including a synovial herniation in ligament fibers, the ectopic inclusion of synovial tissue, a posttraumatic connectival degeneration and, finally, the proliferation of totipotent mesenchymal cells. From a histologic point of view, "synovial ganglion" is a much better definition than "synovial ganglion cyst", because the typical wall of real synovial cysts is missing. The MR patterns are typical of the morphological features described and of the presence of high protein fluid content.

Adult↗
PubMed1995

[Clinical usefulness of a computerized tomography study of morphology of the femoral canal in patients candidates for hip prosthesis].

In the last few years hip arthroplasty has been increasingly used and both metaphysis and diaphysis of the proximal femur are studied preoperatively for best compliance between prosthesis and bone. Indeed, the best results can be obtained by reducing the risk of stem end mobilization, which means to choose the prosthesis fitting the femoral canal best and to limit the use of cement prostheses, which are at high risk of mobilization in time, to advanced osteoporosis patients. We used a simple and repeatable CT technique to study femoral canal structure and size. After accurately positioning the patients inside the gantry, we acquired some axial scans at scheduled levels referring to the horizontal midline of the lesser trochanter. The axial scans were acquired 2 cm above and 5 and 10 cm below the horizontal midline in 105 patients. Measurements were bilateral in 13 patients. Finally, the results were compared with the surgical outcome. Femoral canal cross-diameters and cortical bone width varied greatly, which variations were confirmed in bilateral measurements and in the same patient between the two femurs. Femoral canal structure, which can be remodelled, influences the choice of the stem and thus the amount of cement. We believe this method to be fundamental for correct surgical planning and for best treatment outcome.

Female↗
PubMed1994

Intracellular dynamics of ricin followed by fluorescence microscopy on living cells reveals a rapid accumulation of the dimeric toxin in the Golgi apparatus.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.

Animals↗
PubMed1994

Cyanide dissociation from the hemoglobin of Parascaris equorum.

The reduction of cyanomethemoglobin by dithionite leads to the appearance of an intermediate, the complex of cyanide with ferrous hemoglobin, whose dissociation is easily followed in a stopped flow apparatus. This reaction was studied in the hemoglobin from the parasitic nematode Parascaris equorum, whose extremely high oxygen affinity is due to a very low dissociation rate. The rate of cyanide dissociation from ferrous Parascaris hemoglobin is not so dramatically different from that of other hemoglobins and myoglobins. Other features of the reaction are: (i) the rate constant of cyanide release is pH independent, an observation which is agreement with the possible absence of the distal histidine, given the mechanism suggested in a previous study (Bellelli, A., Antonini, G., Brunori, M, Springer, B.A. and Sligar, S.G. (1990) J. Biol. Chem. 265, 18898-18901), and (ii) the time-course shows no kinetic cooperativity. The structural basis of the extremely high oxygen affinity of Parascaris hemoglobin cannot be explained on the basis of the results here reported. This study also confirms that, even though cyanide binding to ferrous hemoglobins is controlled by distal interactions, the functional behaviour of this ligand is characteristic and differs from the behaviour of oxygen.

Allosteric Regulation↗
PubMed1994

Analysis of biochemical processes in single living cells by quantitative microscopy.

Spectroscopic measurements in single living cells are made possible by the development of computer controlled light detectors, which, when applied to optical microscopes, yield spatial, temporal and eventually spectroscopic information about the sample. This minireview describes some experiments in which the distribution and concentration of specific intracellular markers (proteins, protein complexes, RNA) has been followed by quantitative microscopy. The examples chosen have contributed to shed light on a biochemical process as it happens in vivo; because of the non ideal conditions of the intracellular milieu, the comparison of in vivo and in vitro experiments is of great relevance to the understanding of cellular physiology.

Animals↗
PubMed1993

Ligand binding and slow structural changes in chlorocruorin from Spirographis spallanzanii.

Chlorocruorin is a cooperative respiratory pigment found in the blood of polychaete worms; its prosthetic group is a derivative of the iron protoporphyrin IX, in which the vinyl group at position 2 is substituted by a formyl group. The quaternary structure of chlorocruorins is complex: myoglobin-like subunits are grouped in tetramers and tetramers in dodecamers; 12 dodecamers are assembled in the 3500-kDa particle. Chlorocruorin from Spirographis spallanzanii displays the following overall functional properties: (i) the oxygen affinity is lower than in human hemoglobin, while that of CO is similar if not higher; (ii) the rates of combination with oxygen and carbon monoxide are low; and (iii) the off rate of oxygen is comparable to that of human hemoglobin, while the off rate of CO is 10 times smaller. When CO is partially photolyzed with a long and powerful light flash (70 microseconds), rebinding is biphasic as in mammalian hemoglobins; however, the slowest rate is faster than that observed by stopped flow, suggesting that the unliganded protein decays from the liganded high affinity state (R) to an intermediate state before reaching the low affinity (T) state. Oxygen binding was followed by stopped-flow and flash photolysis. While partial photolysis yields a fast, second-order time course, stopped-flow experiments yield slow, biphasic, and non-second-order time courses. This pattern of reactivity was attributed to a slow conformational transition(s) which is (are) rare limiting with oxygen, but not with CO.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Hydration and allosteric transitions in hemoglobin.

Sucrose and other neutral solutes reduce the oxygen affinity of human hemoglobin. This effect was attributed by Colombo et al. (Colombo, M.F., Rau, D.C., and Parsegian, V.A. (1992) Science 256, 655-659) to a stabilization of the deoxy-T quaternary state of hemoglobin A, via a reduction of the activity coefficient of water. This was correlated to crystallographic results which showed that a significant surface area at the alpha 1 beta 2 interface, which is exposed to the solvent in the oxy-R state, is buried in the deoxy-T state. We show that sucrose has no effect on the oxygen affinity of trout hemoglobin I, which is cooperative in oxygen binding but lacks heterotropic effects, and that in spite of the large buried surface exposed to solvent upon dissociation of human hemoglobin into alpha beta dimers, sucrose leads either to an increased dissociation of hemoglobin A-CO into dimers or to no effect at all (in the presence of inositol hexakisphosphate). These results may demand a reconsideration of the hypothesis extensively discussed by Colombo et al.

Allosteric Regulation↗
PubMed1992

Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.

Allosteric Regulation↗
PubMed1992

Ligand binding by the chlorocruorin from Eudistylia vancouverii.

Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.

Animals↗
PubMed1992

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.

Animals↗