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Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 163 records · Page 9Linked to original sources
PubMed1991

Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography.

A method for a simultaneous separation of malondialdehyde (MDA), ascorbic acid and adenine nucleotide derivatives in biological samples by ion-pairing high-performance liquid chromatography is presented. The separation is obtained by an LC-18-T 15 cm x 4.6 mm 3 microns particle size column using tetrabutylammonium as the pairing ion. The starting buffer consists of 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 plus 1% methanol, pH 7.00. A step gradient is formed using a second buffer consisting of 2.8 mM tetrabutylammonium hydroxide, 100 mM KH2PO4 plus 30% methanol, pH 5.5. Under these chromatographic conditions a highly resolved separation of MDA, ATP, ADP, AMP, adenosine, ascorbic acid, GTP, GDP, IMP, inosine, Hypoxanthine, Xanthine, uric acid, NAD, and NADP can be performed in about 36 min. In addition, the separation of NADH and NADPH can also be obtained; this renders the present method suitable for the detection of these reduced coenzymes in alkaline extracts from tissue samples. Data referring to PCA extracts from ischemic and reperfused isolated rat hearts and from human erythrocytes peroxidized in vitro by a challenge with 1 mM NaN3 and various concentrations of H2O2 are reported. The relevance of this chromatographic method lies in the possibility to determine directly MDA concentrations avoiding the unspecific thiobarbituric acid colorimetric test, any other manipulation of the sample out of the PCA extraction, and any possible coelution of other acid soluble compounds. The simultaneous determination of MDA, ascorbic acid, and of ATP and its degradation products gives the opportunity to correlate, by a single chromatographic run, peroxidative damages with the energy state of the cell which is of great importance in studies of ischemic and reperfused tissues.

Adenine Nucleotides↗
PubMed1991

Lobster haemocyanin. Influence of acclimatization on subunit composition and functional properties.

Haemocyanin from the lobster Palinurus elephas has been shown to change in its subunit composition according to the time of year. In contrast, in Palinurus mauritanicus, a lobster living at greater depth, no seasonal changes in subunit composition have been observed. The results obtained from a set of experiments performed on some Palinurus mauritanicus acclimatized in an aquarium have clearly indicated that modifications of haemocyanin subunit composition may be involved in the adaptation of arthropods to environmental change.

Acclimatization↗
PubMed1991

Temperature modulation of bovine hemoglobins.

The functional properties of hemoglobin from Egyptian water buffalo have been characterized as a function of pH, temperature and chloride concentration. Alongside overall similarities shared with ox and Arctic ruminant hemoglobins, hemoglobin from buffalo shows significant differences with respect to the effect of temperature. The results obtained may suggest that the limited effect of temperature on oxygen binding recently reported for ox hemoglobin could be regarded as an interesting case of a reminiscence of a past glacial age.

Animals↗
PubMed1991

The primary structure of hemoglobin from reindeer (Rangifer tarandus tarandus) and its functional implications.

The primary structures of alpha- and beta-chains of hemoglobin from reindeer (Rangifer tarandus tarandus) were determined. Comparison of the reindeer hemoglobin sequence with those of human and bovine hemoglobins showed 50 and 29 substitutions per alpha beta dimer, respectively. The influence of replacements on the modulation of hemoglobin oxygen affinity by heterothopic ligands and temperature, as well as their importance on the structure-function relationships in hemoglobin are discussed.

Amino Acid Sequence↗
PubMed1991

A new monoclonal antibody to an age sensitive band 3 transmembrane segment.

The appearance of band 3 structural modifications related to aging could be evidenced by means of monoclonal antibodies against senescence antigen. Hence in the attempt to provide an immunological marker of erythrocyte aging, we raised a monoclonal antibody against native band 3 (B6 MoAb), which seems to detect differences in the band 3 molecule from erythrocytes of different ages separated by density gradient. Densitometric evaluation of immunoblotting patterns indicates that the in vivo aging is associated with band 3 monomer degradation. The Percoll separated fractions show a significant increase of those proteolytic fragments that bind the B6 antibody. Finally, protease digestions of unsealed membrane ghosts have been performed to test the binding site of the B6 antibody on the band 3 molecule. The data show that the B6 antibody binds a 19 KDa chymotryptic-tryptic fragment which corresponds to a segment of the looped membrane domain whose steric structure appears to be sensitive to age.

Animals↗
PubMed1991

Effect of exogenous fructose-1,6-bisphosphate on glycolysis in the isolated perfused rat heart.

To test the mechanism of action of fructose-1,6-bisphosphate (F-1,6-P2), experiments were conducted on isolated perfused rat hearts to measure the glycolytic rate supported by exogenous glucose with simultaneous measurement of oxygen consumption and the release of lactate and pyruvate. Glycolysis was assayed in terms of the release of tritiated water from [5-3H] glucose, a measure of the rate through the aldolase step. It was found that 5 mmol/L F-1,6-P2 reduced the glycolytic rate parallel to the decrease in oxygen consumption. The results suggest that the cardioprotective action of F-1,6-P2 is related to a substrate effect and a decrease in adenosine triphosphate consumption as indicated by a decrease in oxygen consumption in accordance with the recent demonstration of Ca2+ binding by F-1,6-P2.

Animals↗
PubMed1991

Preserving effect of fructose-1,6-bisphosphate on high-energy phosphate compounds during anoxia and reperfusion in isolated langendorff-perfused rat hearts.

Isolated Langendorff-perfused rat hearts after 10 min pre-perfusion, were subjected to a substrate-free anoxic perfusion (20 min) followed by 20 min reperfusion with a glucose-containing oxygen-balanced medium. A similar experimental protocol was repeated in the presence either of 5 mM fructose or of 5 mM fructose-1,6-bisphosphate throughout the different perfusion conditions. High-energy phosphate compounds (adenosine triphosphate, creatine phosphate), adenine nucleotides, nicotinic coenzymes, lactate, pyruvate and glycogen content in the tissue were determined at the end of each perfusion period, while coronary flow, heart rate and lactate and pyruvate output were monitored throughout the whole duration of the experiments. On the whole, the results indicate that exogenous fructose-1,6-bisphosphate preserves high-energy metabolites during anoxia and restores myocardial metabolism and contractility during reperfusion, that a prolonged period of substrate-free anoxic perfusion renders the heart unable to normalize its metabolism during re-oxygenation and that fructose is not utilized by the heart for its energy demand. A possible hypothesis concerning the mechanism of action of fructose-1,6-bisphosphate is presented.

Adenine Nucleotides↗
PubMed1991

Oxygen transport in extreme environments.

Evolution has adopted different strategies to solve the problem of transporting oxygen to respiring tissues, according to needs dictated by the environment. A thermodynamic analysis of haemoglobins of organisms living in extreme polar environments (mammals and fish) provides elegant examples of such adaptations.

Adaptation, Physiological↗
PubMed1991

Mechanism of the effect of exogenous fructose 1,6-bisphosphate on myocardial energy metabolism.

The effects of fructose 1,6-bisphosphate (F-1,6-P2) on the isolated Langendorff-perfused heart were studied by monitoring flavoprotein fluorescence, oxygen consumption (MVO2), coronary flow (Fc), systolic intraventricular pressure (Psys), diastolic intraventricular pressure, and contraction frequency. The cellular energy state and cytosolic pH were determined by means of 31P nuclear magnetic resonance. Infusion of 5 mM F-1,6-P2 caused a rapid shift toward reduction in the flavoprotein redox state and initial 50% and 44% decreases in Psys and MVO2, respectively. After a partial recovery, these measures remained 11% and 25% below the basal value. Concomitantly, after an initial transient increase of 13%, Fc remained 17% lower than in the basal state. When the F-1,6-P2 concentration was subsequently increased to 10 mM, psys and MVO2 dropped temporarily to 31% and 29% of the basal value and then remained at 50% and 53%, respectively. Simultaneously, a brief increase was observed in Fc, which then fell 34% below the basal value. Rapid reoxidation of the flavoproteins and increases in MVO2, Psys, and Fc occurred on discontinuation of the F-1,6-P2 infusion. 31P nuclear magnetic resonance during infusions of both 5 and 10 mM F-1,6-P2 revealed a decrease in cytosolic inorganic phosphate and a tendency to increase creatine phosphate, suggesting elevation in the cellular energy state. No changes in intracellular pH occurred as estimated from the chemical shift of the nuclear magnetic resonance of inorganic phosphate. F-1,6-P2 (5 mM and 10 mM) lowered the free Ca2+ concentration in the Krebs-Henseleit bicarbonate buffer (by 32% and 47%, respectively). This probably explains the effects of F-1,6-P2 on mechanical work performance and cellular respiration. A direct metabolic effect also exists, however, because flavoprotein reduction by F-1,6-P2 could be observed in the K(+)-arrested heart, where its effects on MVO2 were minimal. This redox effect may not be caused by changes in free Ca2+ concentration because it could not be reproduced by infusion of EGTA.

Animals↗
PubMed1991

Monoclonal antibodies against protease inhibitors: the case of the bovine pancreatic trypsin inhibitor.

A monoclonal antibody (MoAb) directed against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a small protein made up by 58 aminoacids, has been produced. Mice were immunized with the native form of the protein and gave low antibody response. After somatic hybridization of spleen cells from immunized mice, few clones secreting antibodies against BPTI have been found and just two of them were stable. Both secreted IgM. One (ICI) produces a monoclonal antibody which binds to BPTI with an equilibrium dissociation constant, Kd, of 6.1 x 10(-7) M at pH 7.4. Competition experiments demonstrated that ICI recognizes an epitope close to the reactive site of BPTI. Furthermore, Kd of ICI with an isoinhibitor similar to BPTI (S.I. II) but with few differences at the active site is higher, confirming specificity of binding.

Animals↗
PubMed1991

Antibodies against small molecules.

Compounds with low molecular weight (haptens) are not usually immunogenic. Thus, an hapten should be conjugated with a carrier protein therefore immunizing an animal, in order to induce a strong immune response and in turn obtain large amount of antibodies. Suitable macromolecules are usually albumins, thyroglobulins, haemocyanins and polylysine. The production of specific antibodies against haptens is important both for assays of hormones and drugs in biological fluids or for therapeutic applications in tumour therapy. Approaches in choice of both carrier protein and conjugation methods will be described.

Animals↗
PubMed1990

Interaction of hemoglobin with chloride and 2,3-bisphosphoglycerate. A comparative approach.

The equilibrium oxygen-binding properties of hemoglobins from reindeer (Rangifer tarandus tarandus), musk ox (Ovibos muschatos) and a bat (Rousettus aegyptiacus) have been investigated with special reference to the effect of heterotrophic ligands such as chloride and 2,3-bisphosphoglycerate [Gri(2,3)P2]. The results obtained with hemoglobins from reindeer and musk ox indicate that their low oxygen affinity and their insensitivity to Gri(2,3)P2 are not only an intrinsic property of the molecule, as proposed in the case of ruminant hemoglobins, but also the results of the interplay between chloride and Gri(2,3)P2 interactions. In other words, insensitivity of reindeer and musk ox hemoglobins to Gri(2,3)P2 is mainly due to a decreased affinity constant for this cofactor and to an increased affinity constant for chloride anions; this renders more effective the competition of chloride for th anion-binding site. On the other hand bat hemoglobin behaves in a completely different way and could be regarded as a type case of low-affinity hemoglobin since its functional properties are modulated neither by chloride nor by Gri(2,3)P2. The results are discussed in the light of the amino acid residues which are known to be involved in the binding of organic phosphates.

Allosteric Regulation↗
PubMed1990

Temperature modulation of oxygen transport in a diving mammal (Balaenoptera acutorostrata).

The functional properties of haemoglobin from the Lesser Rorqual whale (Balaenoptera acutorostrata) have been characterized as a function of the heterotropic effector concentrations and temperature. The results obtained suggest the existence of sophisticated modulation mechanisms based on the interplay of organic phosphates, carbon dioxide, lactate and temperature. These, together with the very small apparent heat of oxygenation (delta H) of oxygen binding, have been physiologically interpreted on the basis of the specific metabolic needs of this diving mammal.

2,3-Diphosphoglycerate↗
PubMed1990

Flight and heat dissipation in birds. A possible molecular mechanism.

Birds during normal sustained flight must be able to dissipate more than 8 times as much heat as during rest in order not to be overheated. The experiments reported in this note on the hemoglobin systems from two different birds indicate the existence of a molecular mechanism by which hemoglobin is used simultaneously for oxygen transport and heat dissipation.

Animals↗
PubMed1990

Thermodynamics of oxygen binding to arctic hemoglobins. The case of reindeer.

The most surprising characteristic of reindeer hemoglobin (Hb) concerns its response to changes in temperature. Thus, the shape of the oxygen-binding curve is strongly temperature dependent due to the difference in the enthalpy of oxygenation between the T and R state of the molecule. In fact, delta H of oxygen binding to the T state is strongly exothermic whereas that of the R state is very close to zero or possibly positive after correction for the heat of oxygen solubilization. Moreover, the allosteric transition T0----R0 has been found to display a negative delta H and a contemporaneous decrease in entropy, a behavior which is precisely the opposite of what has been reported for other hemoglobins. As a whole, reindeer Hb represents a beautiful example of the significance that comparative studies may have in assessing the general validity of the main properties of the hemoglobin molecule.

2,3-Diphosphoglycerate↗
PubMed1990

Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.

Despite the fact that the horse is one of the more common domesticated animals, there are few reports dealing with the properties of its blood, and no comprehensive study has been performed on the reactivity of horse haemoglobin towards organic and inorganic ions. Here we report data on the effects of the organic phosphates D-glycerate-2,3-bisphosphate (2,3-DPG) and InsP6, and of chloride on the properties of horse haemoglobin. Thus the effect of saturating concentrations of 2,3-DPG on the oxygen affinity of horse haemoglobin is about 60% lower than with human adult haemoglobin under the same experimental conditions. The same applies also to InsP6, whose effect on oxygen binding to horse haemoglobin is decreased by about 55% compared with human adult haemoglobin. On the whole, horse haemoglobin appears to be much less sensitive to organic phosphates than previously believed. These results are discussed in the light of the primary structure of the molecule.

2,3-Diphosphoglycerate↗
PubMed1990

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia.

The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range -0.44- -0.73. The Bohr effect is more pronounced at 20 degrees C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, delta H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.

Acclimatization↗