Biomedical subjects

M Coletta

Publications and source records attributed to M Coletta.

At least 73 records · Page 4Linked to original sources

PubMed1994

Synergistic modulation by chloride and organic phosphates of hemoglobin from bear (Ursus arctos).

The oxygen binding properties of hemoglobin (Hb) from brown bear (Ursus arctos) have been studied focussing on the effect of heterotropic ligands, and the behaviour has been compared with that of human HbA, taken as a prototype of mammalian Hbs. It has been observed that in bear Hb chloride ions and 2,3-diphosphoglyceric acid (Gri(2,3)P2) can modulate the oxygen affinity in a synergistic way such that their individual effect is enhanced whenever they are both present in saturating amounts. The thermodynamic analysis of such a feature indicates that in bear Hb there are two classes of chloride binding sites, one acting synergistically with Gri(2,3)P2 and another one, which likely overlaps with the organic phosphate interaction cleft, and is therefore fully operative only in the absence of Gri(2,3)P2. The behaviour of the last site is similar to that observed in human HbA, where the effect of Cl- and Gri(2,3)P2 is mutually exclusive. The interaction energy between chloride and Gri(2,3)P2 synergistic binding sites appears to be O2-linked so that the interplay may have a relevant physiological role in modulating the oxygen transport in brown bear. This behaviour is associated with a marked pH-dependence of the oxygenation enthalpy in bear Hb, such that under acidotic and hypercloruremic conditions, oxygen supply to peripheral tissues could be maintained essentially unaltered even under low temperature conditions.

Animals↗

PubMed1994

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a spectroscopic and thermodynamic study.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated quantitatively by EPR and absorbance spectroscopy. The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and baker's yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins. Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x 10(-6) M and < or = 5 x 10(-8) M, respectively, at pH 7.0 and 10 degrees C. The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-1, at 10 degrees C).

Animals↗

PubMed1994

Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis.

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers first appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

A polymerising Root-effect fish hemoglobin with high subunit heterogeneity. Correlation with primary structure.

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the alpha and beta chain has been established. The presence of a reactive Cys in the external position beta CD8(49) causes polymerisation through intermolecular disulfide bridges between beta chains, with no alteration of functional features. C. kumu Root-effect hemoglobin displays very low or no subunit co-operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH-dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co-operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pKa of the proton-linked effect on the slow-reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by Ile. Although this residue has been shown not to significantly alter ligand binding to the alpha chain, to some extent it can perturb the access of oxygen to the beta chain. Thus, this substitution may be the main reason for subunit functional heterogeneity.

Adenosine Triphosphate↗

PubMed1993

Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex).

The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals.

Amino Acid Sequence↗

PubMed1993

Paracrine stimulation of senescent satellite cell proliferation by factors released by muscle or myotubes from young mice.

The proliferative potential of satellite cells undergoes a dramatic decrease in the early postnatal period and a more modest but continuous decrease throughout the life span of the animal. To address the problem of the mechanism regulating this phenomenon and to understand whether it is causally linked to senile muscle atrophy, we studied the response of aged satellite cells to serum and to different growth factors. The data reported indicate a generalised reduction in the response to all mitogens tested, which could not be compensated for by increased concentrations of serum or growth factors. On the other hand, conditioned medium of differentiated myotubes from young mice exhibited a strong mitogenic action on aged satellite cells, while conditioned media of myotubes from old mice or from a variety of non-muscle cells were ineffective. Furthermore, saline extracts from muscle of young mice are also able to exert this mitogenic action. Saline extracts of muscle from old mice were poorly mitogenic for satellite cells from young mice, and not at all for satellite cells from old mice. These data indicate that paracrine interactions operate inside the muscle tissue and are probably required for the normal replicative behaviour of satellite cells. The failure of such interactions may be among the causes leading to age-related muscle hypotrophy.

Animals↗

PubMed1993

Interaction of inositol hexakisphosphate with liganded ferrous human hemoglobin. Direct evidence for two functionally operative binding sites.

Inositol hexakisphosphate (InsP6) binding to the oxygenated, carbonylated and nitrosylated derivatives of ferrous human hemoglobin (HbO2, HbCO and HbNO, respectively) has been measured at pH 7.0 (0.1 M Bis-Tris buffer, 0.1 M NaCl) and 20 degrees C. The observations indicate the presence of two InsP6 binding sites per tetramer in all the heme liganded hemoglobin derivatives, with different affinities for the polyphosphate. For each binding site, InsP6 interacts with similar affinity constants to HbO2, HbCO and HbNO. Such a finding indicates that different heme ligands do not alter significantly the stereochemistry of the polyphosphate binding cleft. This behaviour seems to indicate that, even though different heme ligands are likely to affect the tertiary conformation of the subunit in a different fashion, the perturbation does not seem to be transmitted to the quaternary arrangement of the whole macromolecule, and, thus, to the InsP6 binding site.

Binding Sites↗

PubMed1993

Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 x 10(-1) M N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 x 10(-1) M NaCl), as well as at 100 K and/or 20 degrees C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.

Bezafibrate↗

PubMed1993

Retroviral vector-mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo.

Primary human myogenic cells isolated from fetal and adult muscle were infected with a high-titer, Moloney murine leukemia virus (MoMLV)-derived retroviral vector expressing a bacterial beta-galactosidase (beta-gal) gene under long terminal repeat (LTR) control. Gene transfer efficiency averaged 50% in both fetal myoblasts and adult satellite cells, as revealed by beta-gal staining. The reporter gene was stably integrated, faithfully inherited, and expressed at significant levels in myogenic cells for at least 10 generations under clonal growth conditions, and throughout the culture life span upon differentiation into myotubes. Comparable gene transfer efficiency was obtained in myogenic cells from muscle biopsies of patients affected by a number of genetic or acquired myopathies, including Duchenne muscular dystrophy. Transduced normal human satellite cells were injected into regenerating muscle of immunodeficient mice, where they formed new muscle fibers in which the product of the reporter gene was detectable for 2 months after injection. These results show that retroviral vectors can be used to transfer foreign genes with high efficiency into normal or abnormal primary human myogenic cells, leading to stable expression into mature muscle. Satellite cells engineered in this way might represent an effective tool for gene therapy of muscular dystrophies as well as for systemic delivery of recombinant gene products for correction of inherited and acquired disorders. The human-mouse model described here will allow in vivo testing of such gene therapy approaches.

Adolescent↗

PubMed1993

Binding of bovine basic pancreatic trypsin inhibitor (Kunitz) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human cathepsin G: a kinetic and thermodynamic study.

The effect of pH and temperature on kinetic and thermodynamic parameters for the binding of the bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor; BPTI) as well as bovine and porcine pancreatic secretory trypsin inhibitor (Kazal inhibitor; bovine and porcine PSTI, respectively) to human cathepsin G (EC 3.4.21.20) has been investigated. The affinity of the macromolecular inhibitors examined for cathepsin G is characterized by an endothermic, entropy-driven, behaviour, and shows the following trend: BPTI > bovine PSTI > porcine PSTI. The affinity difference of BPTI as well as of bovine and porcine PSTI for cathepsin G is mostly accounted for by changes in the values of the apparent dissociation rate constant for the proteinase:inhibitor complex destabilization. On increasing the pH from 4.5 to 9.5 (at 25.0 degrees C), the affinity of BPTI, as well as bovine and porcine PSTI for cathepsin G increases thus reflecting the acidic-pK shift of the His-57 catalytic residue from approximately 6.9 in the free enzyme to approximately 5.0 in the serine proteinase:inhibitor complexes. The BPTI as well as the bovine and porcine PSTI binding properties of cathepsin G have been analyzed in parallel with those of related serine (pro)enzyme/macromolecular inhibitor systems. Considering the known molecular models, the observed binding behaviour of BPTI as well as that of bovine and porcine PSTI to cathepsin G has been related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).

Animals↗

PubMed1993

Structural studies on the loggerhead sea turtle (Caretta caretta) myoglobin.

The primary structure of myoglobin from the loggerhead sea turtle (Caretta caretta) has been determined; the protein consists of 153 amino acid residues. The ferric loggerhead sea turtle myoglobin has been crystallized in a form suitable for X-ray structural investigations. The crystals were grown at pH 8.0, in 0.05 M tris/HCl buffer, using 3.2 M ammonium sulfate as precipitating agent, at 4 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell constants a = 37.2 A, b = 61.1 A, c = 75.2 A (one molecule, 17,000 M(r), in the asymmetric unit). A molecular replacement solution was found for the loggerhead sea turtle myoglobin crystals using sperm whale myoglobin structure as search model. The R-factor value, after molecular replacement, is 0.387, for the data in the 15-3.3 A resolution range. The results here reported are the basis for the first X-ray crystallographic investigation on a reptile myoglobin, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian (i.e. sperm whale) myoglobins.

Amino Acid Sequence↗

PubMed1992

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.

Animals↗

PubMed1992

Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).

2,3-Diphosphoglycerate↗

PubMed1992

Structural study and preliminary crystallographic data for the hemoglobin from reindeer (Rangifer tarandus tarandus)

The ferric form of reindeer hemoglobin (Rangifer tarandus tarandus) has been crystallized in an orthorhombic crystalline form from polyethylene glycol solutions, at pH 8.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell edges a = 84.2 A, b = 59.9 A, c = 119.5 A; one hemoglobin tetramer is contained in the asymmetric unit. The crystals diffract X-rays to a limit spacing of 3.0 A. Inspection of amino acid sequences in the N-terminal region of beta-chains, and analysis of hemoglobin three-dimensional models, allows one to rationalize, on a molecular basis, the reduced O2 affinity and the decreased effect of organic phosphates observed in ruminant hemoglobins. By analogy, the analysis is extended to birds and reptiles, whose hemoglobin beta-chains display, as in ruminants, the deletion of the N-terminal residue and a methionine at the NA2 position.

2,3-Diphosphoglycerate↗

PubMed1992

Binding of hirudin to human alpha, beta and gamma-thrombin. A comparative kinetic and thermodynamic study.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

Aprotinin↗

PubMed1992

A comparative study of the temperature dependence of the oxygen-binding properties of mammalian hemoglobins.

The effect of temperature on the oxygen-binding properties of hemoglobin (Hb) from ruminants, such as ox, reindeer, musk ox, mouflon and egyptian water buffalo is compared to that of human adult Hb (HbA). A striking difference emerges where in the presence of chloride ions and in the absence of 2,3-diphosphoglycerate [Gri(2,3)P2] a strongly reduced exothermic oxygenation process is observed for all ruminant Hb investigated with respect to HbA. Next, in the presence of physiological concentrations of Gri(2,3)P2, HbA displays a less exothermic oxygenation process, with values tending toward those observed in ruminant Hb [where Gri(2,3)P2 is not a physiological effector and for which the addition of Gri(2,3)P2 has essentially no effect on the oxygenation enthalpy]. Different from HbA, the intrinsically less exothermic oxygen binding seems to be independent of the experimental conditions for ruminant Hb, underlying specific structural characteristics which might be responsible for this feature.

2,3-Diphosphoglycerate↗

PubMed1992

The 'natural' hybrid haemoglobin from mule. Interrelationships with its parent haemoglobins from horse and donkey.

The equilibrium O2-binding properties of the hybrid haemoglobin (Hb) present in vivo in erythrocytes from mule and of its parent Hbs from horse and donkey were compared with special reference to the effect of heterotropic ligands such as Cl-, D-glycerate 2,3-bisphosphate (DPG) and inositol hexakisphosphate. All these Hbs display a decreased effect by polyphosphates, confirming that what has been observed for horse Hb [Giardina, Brix, Clementi, Scatena, Nicoletti, Cicchetti, Argentin & Condò (1990) Biochem. J. 266, 897-900] is common to other equine species, at least from a qualitative standpoint. However, different quantitative aspects can be detected, which can be accounted for by a different role for the two types of chain in characterizing the binding free energy for the various heterotropic effectors. In particular, it is shown that the binding mode of DPG and inositol hexakisphosphate displays different features since long-range effects can be observed clearly for inositol hexakisphosphate but not for DPG. In general terms, in spite of a different intrinsic O2 affinity, the modulation of functional properties by third ligands leads these Hbs to behave, under physiological conditions, similarly to human HbA. It might represent an interesting example of how different species with similar functional needs find different ways to produce a similar functional behaviour.

Animals↗

PubMed1992

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis. Kinetic and spectroscopic evidence.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

Animals↗