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Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 127 records · Page 7Linked 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

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Animals↗
PubMed1994

Time dependence of plasma malondialdehyde, oxypurines, and nucleosides during incomplete cerebral ischemia in the rat.

Incomplete cerebral ischemia (30 min) was induced in the rat by bilaterally clamping the common carotid arteries. Peripheral venous blood samples were withdrawn from the femoral vein four times (once every 5 min) before ischemia (0 time) and 5, 15, and 30 min after ischemia. Plasma extracts were analyzed by a highly sensitive high-performance liquid chromatographic method for the direct determination of malondialdehyde, oxypurines, and nucleosides. During ischemia, a time-dependent increase of plasma oxypurines and nucleosides was observed. Plasma malondialdehyde, which was present in minimal amount at zero time (0.058 mumol/liter plasma; SD 0.015), increased after 5 min of ischemia, resulting in a fivefold increase after 30 min of carotid occlusion (0.298 mumol/liter plasma; SD 0.078). Increased plasma malondialdehyde was also recorded in two other groups of animals subjected to the same experimental model, one receiving 20 mg/kg b.w. of the cyclooxygenase inhibitor acetylsalicylate intravenously immediately before ischemia, the other receiving 650 micrograms/kg b.w. of the hypotensive drug nitroprusside at a flow rate of 103 microliters/min intravenously during ischemia, although in this latter group malondialdehyde was significantly higher. The present data indicate that the determination of malondialdehyde, oxypurines, and nucleosides in peripheral blood, may be used to monitor the metabolic alterations of tissues occurring during ischemic phenomena.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Free radical production by activated haem proteins: protective effect of coenzyme Q.

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (MbIV and/or HbIV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (MbIV and/or HbIV) back to met and oxy forms (Mb and/or HbIIO2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

Animals↗
PubMed1994

Lipids and lipoproteins in acute lymphoblastic leukaemia (ALL).

Current studies suggest a derangement of the cholesterol homeostasis in certain types of leukaemia. The low serum cholesterol levels observed in patients with AML have been related to the tumoral mass and to the disease activity. Only a few studies have taken into consideration a possible change of the serum lipids in ALL. The aim of this study was to evaluate a possible prognostic significance of serum levels of total cholesterol (TC), triglycerides, HDL-C, LDL-C, VLDL-C, apolipoproteins A1 and B and lipoprotein (a), investigated in 10 newly diagnosed ALL patients before and after induction treatment. At diagnosis all these parameters were not significantly different in our patients vs control group except cholesterol and HDL-C levels, which were significantly lower. After the induction treatment, we found a significant increase of HDL-C and Apo A1 values only in those patients that achieved a complete remission. These results support the idea that some serum lipids, such as HDL-C and Apo A1, may have a role as early and reliable markers of the effectiveness of chemotherapy.

Adult↗
PubMed1994

Myocardial release of malondialdehyde and purine compounds during coronary bypass surgery.

BACKGROUND: Free radicals and lipid peroxidation have been suggested to play an important role in the pathophysiology of myocardial reperfusion injury. The purpose of the present study was to monitor myocardial malondialdehyde (MDA) production as an index of lipid peroxidation during ischemia-reperfusion sequences in patients undergoing elective coronary bypass grafting. There has been a lot of debate on the role of xanthine oxidase as a potential superoxide anion generator and thus lipid peroxidation in human myocardium. To evaluate the activity of xanthine oxidase pathway, we measured the changes in the transcardiac concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid. METHODS AND RESULTS: The coronary sinus-aortic root differences (CS-Ao) of MDA, oxypurines, and nucleosides were measured by a recently developed ion-pairing high-performance liquid chromatographic (HPLC) method. Fifteen patients were included in the study, and 13 of them demonstrated a more than 10-fold increase in net myocardial production of MDA on intermittent reperfusion during the aortic cross-clamp period. In 2 patients, MDA was not detectable in any of the CS or Ao samples. Before aortic cross-clamping, the CS-Ao concentration differences in adenosine, inosine, hypoxanthine, xanthine, and uric acid were 0.59 +/- 0.19, 0.23 +/- 0.05, 0.89 +/- 0.36, 0.58 +/- 0.32, and 11.4 +/- 4.9 mumol/L, respectively. After aortic cross-clamping, the sum of the transcardiac differences of these compounds increased up to 2.8-fold and then gradually decreased after declamping of the aorta. There was a weak positive correlation between transcardiac concentration differences of MDA and xanthine plus uric acid (r = .48, P < .01). The postoperative functional recovery or leakage of cardiac enzymes was not affected by the level of MDA net release during the aortic cross-clamp period, however. CONCLUSIONS: We conclude that myocardial lipid peroxidation, estimated as MDA formation, is common during intermittent ischemia-reperfusion sequences in coronary bypass surgery, although some patients may be better protected. Xanthine oxidase appears to be operative in human myocardium, and free radicals generated in this reaction might also be involved in the observed lipid peroxidation process. Increased degradation of myocardial adenine nucleotides and concomitant lipid peroxidation may play a specific role in the development of reperfusion injury. In this study, however, more extensive lipid peroxidation was not associated with impaired functional recovery.

Adenosine↗
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

Monoclonal antibody recognizes different quinone moieties in enzymes.

We produced monoclonal antibodies against the coenzyme pyrrolequinoline quinone (PQQ). These antibodies were obtained by immunizing mice with PQQ conjugated to a chemically modified polypeptide in order to induce a strong immune response. Among the various antibodies obtained, one was found to bind (besides PQQ and 6-hydroxydopamine conjugated to carrier proteins) several different quinoenzymes, namely lentil seedling and bovine serum diamine oxidases and methylamine dehydrogenase. This antibody was able to inhibit the catalytic activity of these enzymes. Moreover, the monoclonal antibody recognized different proteins of lentil seeds on Western blots. Even the variable fragment of immunoglobulin heavy chains of this monoclonal antibody expressed in Escherichia coli is able to recognize the active site of different quinoenzymes.

Amine Oxidase (Copper-Containing)↗
PubMed1993

Effectiveness of thrombolysis is associated with a time-dependent increase of malondialdehyde in peripheral blood of patients with acute myocardial infarction.

By using a highly sensitive, high-performance liquid chromatographic technique, plasma values of malondialdehyde (MDA), adenosine and oxypurines were determined in 10 healthy subjects, 10 patients with noncardiac illness, and 20 patients with acute myocardial infarction (AMI) observed within 6 hours from the onset of symptoms. Patients with AMI received fibrinolytic treatment. Peripheral blood was obtained before and serially after thrombolysis (1, 2, 3, 6 and 24 hours). Coronary patency was assessed by timing of peak creatine phosphate kinase and by predischarge angiography. MDA (mean +/- SD) in healthy subjects, noncardiac patients, and immediately before thrombolytic treatment in patients with AMI was 0.051 +/- 0.013, 0.066 +/- 0.020 and 0.397 +/- 0.326 mumol/liter of plasma, respectively. A progressive increase in plasma MDA after thrombolysis was observed only in reperfused patients, whose values at the third, sixth and 24th hours were also significantly greater than those of nonreperfused patients. Time-dependent variations of xanthine and adenosine were also observed in the same group after thrombolysis. The data appear to indicate that a relevant increase in plasma MDA, mostly originating due to phospholipid derangement of postischemic myocytes, occurs only in patients with successful thrombolysis, thus suggesting that if properly assayed, it may represent reliable biochemical evidence of tissue injuries after myocardial reperfusion in humans.

Adenosine↗
PubMed1993

Protein dynamics in minimyoglobin: is the central core of myoglobin the conformational domain?

The kinetics of CO binding to the horse myoglobin fragment Mb-(32-139), the so-called "mini-Mb," were investigated by laser flash photolysis in 0.1 M phosphate buffer and in buffer with 75% (vol/vol) glycerol. The reaction displays complex time courses that can be approximated satisfactorily only with a sum of five exponentials. The features of the kinetic components and a comparison of the deoxy-minus-carbonyl difference spectra of mini-Mb and horse Mb obtained under equilibrium conditions, with the kinetic difference spectra resulting from the global analysis of the traces recorded between 400 and 450 nm, show that CO binding to mini-Mb is accompanied by large structural changes. In view of the fact that mini-Mb is an approximation of the Mb-(31-105) fragment encoded by the central exon of the Mb gene, this finding is particularly relevant. On the basis of our data and previous reports [De Sanctis, G., Falcioni, G., Giardina, B., Ascoli, F. & Brunori, M. (1988) J. Mol. Biol. 200, 725-733; De Sanctis, G., Falcioni, G., Grelloni, F., Desideri, A., Polizo, F., Giardina, B., Ascoli, F. & Brunori, M. (1992) J. Mol. Biol. 222, 637-643], we propose that the protein fragment encoded by the central exon of the Mb gene is the domain responsible for ligand-linked conformational transitions, while the two terminal fragments dampen the amplitude of the structural changes that accompany ligand binding, thus rendering the protein stable and kinetically more efficient in its physiological function.

Animals↗
PubMed1993

Physiological relevance of the overall delta H of oxygen binding to fetal human hemoglobin.

Human fetal hemoglobin is known to display, at 20 degrees C, a lower affinity than human adult hemoglobin for oxygen when both proteins are in the absence of organic phosphates. The physiologically important reverse situation is achieved at 37 degrees C upon addition of 2,3-bisphosphoglycerate (DPG), whose lower effect on fetal hemoglobin is related to some amino acid substitutions present in gamma-chains. However, the difference in oxygen affinity observed at 37 degrees C is not solely due to the different modulation power of DPG with respect to adult and fetal hemoglobins. In fact, the results presented here reveal new aspects linked to the interplay of temperature and organic phosphates. In particular, the lower effect of DPG on fetal hemoglobin renders almost identical the oxygen affinity of the two hemoglobins at 20 degrees C, abolishing the difference observed in the absence of the effector. Successively on going from 20 degrees C to 37 degrees C, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal hemoglobin when in the presence of DPG, adult hemoglobin shows a lower oxygen affinity, as it should if oxygen has to be transferred from maternal to fetal blood.

2,3-Diphosphoglycerate↗
PubMed1993

Antioxidant effect of coenzyme Q on hydrogen peroxide-activated myoglobin.

In recent years increased attention has been focused on the reduced forms of coenzyme Q as antioxidant compounds inhibiting lipid peroxidation in model systems and in biological membranes, but in spite of extensive experimental evidences the molecular mechanisms responsible for the antioxidant activity of ubiquinones are still debated. Ferrylmyoglobin and/or its free radical form are regarded as powerful oxidizing agents capable of promoting oxidation of essential cellular constituents, particularly cell membranes. Therefore, we investigated the effects of ubiquinol on the formation and survival of ferryl species of myoglobin and on metmyoglobin itself. The addition of a threefold molar excess of hydrogen peroxide to a solution of metmyoglobin induces the rapid formation of a compound with the spectral characteristics of ferrylmyoglobin. The reaction is complete within 4 min, producing up to 76% of ferrylmyoglobin, which remains stable for at least 30 min. The addition of ubiquinol-1 to the same solution provokes a rapid and progressive reduction of ferrylmyoglobin to metmyoglobin and oxymyoglobin. Ubiquinol-1, furthermore, is also capable of protecting metmyoglobin against oxidation when added in the solution before hydrogen peroxide. Ubiquinol-1, indeed, is effective at both limiting the maximal ferrylmyoglobin level attained (59% inhibition) and accomplishing complete removal of the ferryl form (in about 15 min). The results demonstrate that ubiquinol is capable of reducing both ferrylmyoglobin and metmyoglobin to oxymyoglobin, providing a novel antioxidant mechanism for coenzyme Q.

Antioxidants↗
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

Activation of 2,4-diaminotoluene to proximate carcinogens in vitro, and assay of DNA adducts.

1. 2,4-Diaminotoluene, which yields adducts with DNA in vivo, has been studied for its ability to form adducts in vitro. Metabolic activation with rat liver post-mitochondrial supernatant gave 300 adducts/10(6) nucleotides in calf thymus single-stranded DNA, under defined experimental conditions. 2. 2,4-Diaminotoluene-modified DNA and deoxyhomopolymers showed characteristic u.v. absorption spectra, exhibiting hyperchromic effects at 235 and 220 nm, and hypochromic effect at 260 nm. The difference spectra between diamine-modified and untreated DNA, or deoxyhomopolymer, were very similar to the spectrum of 2,4-diaminotoluene alone. 3. 2,4-Diaminotoluene-modified DNA was assayed by ELISA with specific monoclonal antibodies directed against diamine-DNA adducts. Reactions with poly-d(A) or poly-d(A-T) gave no spectral modification, and immunochemical analysis showed that the diamine did not bind to these polynucleotides. On the other hand, in the case of poly-d(G) or poly-d(C-G), strong immunoreactions were observed, demonstrating that the guanine base is involved in the binding of the diamine to DNA. 4. Monoclonal antibodies directed against different diamine-DNA adducts have shown that 80% of the in vitro metabolic activation involves the para amino group of the aromatic diamine.

Animals↗
PubMed1992

A monoclonal antibody monitoring band 3 modifications in human red blood cells.

Morphologic and metabolic erythrocyte modifications are thought to be the basis of cell removal from circulating blood. A significant role has been ascribed to the immunological network which may remove aged or misshapen erythrocytes through the binding of specific autoantibodies. Along this line recent observations indicate that a senescence antigen appears in consequence of postsynthetic modifications of band 3, one of the most important erythrocyte membrane proteins, which accounts for many functional activities of the red cells. On this basis, we raised a mouse hybridoma anti-band 3 monoclonal antibody (B6 MoAb) of the IgG2a class which monitors band 3 differences among normal red blood cells separated by Percoll density gradient. These differences are outlined by the decrease of B6 MoAb binding to band 3 monomer, the appearance of an 80-90 kDa new band, lighter than band 3, and the increase of low molecular weight fragments in the 4.5 region. The B6 MoAb appears to be very useful in detecting modifications of band 3 since it bind to a 19 kDa Chy-Try fragment estimated to be sensitive to aging.

Aging↗