Search PubMed⌕ Search

Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 37 records · Page 2Linked to original sources
PubMed2001

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H(2)O(2) (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO(2) (1, 5, 10, 20, and 50 mm), for 15 min at 37 degrees C. AMP-deaminase tremendously increased by increasing H(2)O(2) or NaNO(2) at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5-100 mm), N-ethylmaleimide (0.1-10 mm), or p-hydroxymercuribenzoate (0.1-5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase of V(max), modification of the curve shape of V(o) versus [S], Hill plots, and coefficients) were recorded with 4 mm H(2)O(2)- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe(2+) + H(2)O(2)) or -SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.

AMP Deaminase↗
PubMed2001

A fast chemiluminescent method for H(2)O(2) measurement in exhaled breath condensate.

BACKGROUND: Breath condensate can give useful information on volatile compounds produced at alveolar level. Actual concentration of H(2)O(2) in breath condensate is dependent on its production at alveolar level and on the efficacy of the detoxifying systems, catalase, glutathione peroxidase, etc. METHODS: In the present paper, a simple chemiluminescent method for the determination of the H(2)O(2) collected in exhaled breath is shown and data of both smokers and nonsmokers volunteers are presented. RESULTS: The chemiluminescent response is linear up to 100 micromol/l H(2)O(2). The analytical sensitivity is about 0.01 micromol/l. Most of the nonsmokers have a H(2)O(2) content lower than 0.05 micromol/l, while smokers have a content ranging from 0.1 to 0.6 micromol/l.

Adult↗
PubMed2001

A synthetic peptide corresponding to the 550-585 region of alpha-dystroglycan binds beta-dystroglycan as revealed by NMR spectroscopy.

We have probed the binding of a synthetic peptide corresponding to the region 550-585 of the alpha subunit of dystroglycan with a recombinant protein fragment corresponding to the N-terminal extracellular region of beta-dystroglycan (654-750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra-residue HN-Halpha peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550-565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C-terminal portion is marginally involved. These NMR results have been confirmed by solid-phase binding assays.

Amino Acid Motifs↗
PubMed2001

Role of ergothioneine on S-nitrosoglutathione catabolism.

Ergothioneine (ESH) is a low-molecular-mass thiol present in millimolar concentrations in a limited number of tissues, including erythrocytes, kidney, seminal fluid and liver; however, its biological function is still unclear. In the present study we investigated the role of ESH in the catabolism of S-nitrosoglutathione (GSNO). The results show that: (1) GSNO decomposition is strongly influenced by ESH (k"=0.178+/-0.032 M(-1) x s(-1)); (2) ammonia is the main nitrogen-containing compound generated by the reaction; and (3) nitrite is practically absent under both aerobic and anaerobic conditions. These findings are markedly different from those reported for the GSH-induced decomposition of GSNO, in which the nitrogen-containing end products are nitrite, ammonia and nitrous oxide (N(2)O) under aerobic conditions but nitrite, ammonia, nitric oxide (NO) and small quantities of hydroxylamine under anaerobic conditions. Considering the high concentration of ESH in specific cells, the reaction with GSNO should be considered as an important molecular event occurring in the cell.

Ergothioneine↗
PubMed2001

Molecular dynamics analysis of a second phosphate site in the hemoglobins of the seabird, south polar skua. Is there a site-site migratory mechanism along the central cavity?

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M., A. Riccio, M. Romano, B. Giardina, and G. di Prisco. 2000. Eur. J. Biochem. 267:6089-6098) in the hemoglobins of the south polar skua (Catharacta maccormicki). It is formed by a cluster of six positive charges of both alpha chains (Val-1, Lys-99, Arg-141); the two Lys-99alpha have an essential role in the site structure. The present investigation, carried out on skua deoxyhemoglobins by using a molecular dynamics approach, confirms the structural feasibility of the additional site, possibly having the role of an entry-leaving site, and leads to the proposal of a novel migration pathway for phosphate along the central cavity of hemoglobin from one binding site to the other, occurring according to the hypothesis of a site-site migratory mechanism, which may assign a functional role to the central cavity. The role of Lys-99alpha was further confirmed by molecular dynamics experiments on the mutant Lys-99alpha-->Ala in which, at the end of the simulation, the phosphate was external to the additional site.

Animals↗
PubMed2001

Functional and computer modelling studies of haemoglobin from horse. The haemoglobin system of the Sardinian wild dwarf horse.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr-->Ser) and A2 (Pro-->Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.

Adult↗
PubMed2001

The hemoglobin system of the brown moray Gymnothorax unicolor: structure/function relationships.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.

Allosteric Regulation↗
PubMed2001

Identification of the beta-dystroglycan binding epitope within the C-terminal region of alpha-dystroglycan.

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.

Amino Acid Sequence↗
PubMed2001

Is homocysteine a pro-oxidant?

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

Animals↗
PubMed2001

Human heart cytosolic reductases and anthracycline cardiotoxicity.

Anthracyclines are a class of antitumor drugs widely used for the treatment of a variety of malignancy, including leukemias, lymphomas, sarcomas, and carcinomas. Different mechanisms have been proposed for anthracycline antitumor effects including free-radical generation, DNA intercalation/binding, activation of signaling pathways, inhibition of topoisomerase II and apoptosis. A life-threatening form of cardiomyopathy hampers the clinical use of anthracyclines. According to the prevailing hypothesis, anthracyclines injure the heart by generating damaging free radicals through iron-catalyzed redox cycling. Although the "iron and free-radical hypothesis" can explain some aspects of anthracycline acute toxicity, it is nonetheless disappointing when referred to chronic cardiomyopathy. An alternative hypothesis implicates C-13 alcohol metabolites of anthracyclines as mediators of myocardial contractile dysfunction ("metabolite hypothesis"). Hydroxy metabolites are formed upon two-electron reduction of the C-13 carbonyl group in the side chain of anthracyclines by cytosolic NADPH-dependent reductases. Anthracycline alcohol metabolites can affect myocardial energy metabolism, ionic gradients, and Ca2+ movements, ultimately impairing cardiac contraction and relaxation. In addition, alcohol metabolites can impair cardiac intracellular iron handling and homeostasis, by delocalizing iron from the [4Fe-4S] cluster of cytoplasmic aconitase. Chronic cardiotoxicity induced by C-13 alcohol metabolite might be primed by oxidative stress generated by anthracycline redox cycling ("unifying hypothesis"). Putative cardioprotective strategies should be aimed at decreasing C-13 alcohol metabolite production by means of efficient inhibitors of anthracycline reductases, as short-chain coenzyme Q analogs and chalcones that compete with anthracyclines for the enzyme active site, or by developing novel anthracyclines less susceptible to reductive metabolism.

Anthracyclines↗
PubMed2001

Hyperhomocysteinemia and prevalence of polymorphisms of homocysteine metabolism-related enzymes in patients with inflammatory bowel disease.

OBJECTIVES: Patients with inflammatory bowel disease (IBD) have an increased risk of thrombotic complications. Moreover, a hypercoagulable state has been hypothesized as a contributing factor in the pathogenesis of IBD. Recently, a growing amount of interest has focused on mild-to-moderate hyperhomocysteinemia as a risk factor for thromboembolic disease. We aimed to evaluate the prevalence of hyperhomocysteinemia in patients with IBD and to investigate the contribution of genetic defects in the enzymes involved in homocysteine (Hcy) metabolism and vitamin status in determining increased levels of plasma total Hcy (tHcy). METHODS: The concentrations of tHcy, folate, and vitamin B12 as well as the prevalence of methylenetetrahydrofolate reductase (MTHFR) 677C to T mutation and the 68-bp insertion at exon 8 of cystathionine beta-synthase (CBS) were measured in patients with IBD and healthy controls. RESULTS: In all, 17 out of 64 IBD patients (26.5%) and four out of 121 (3.3%) controls had hyperhomocysteinemia with a statistically significant difference (p < 0.0001). No significant difference was found between IBD patients and controls with regard to the prevalence of homozygotes for the C677T variant (TT) of MTHFR or the prevalence of heterozygotes for the CBS-gene mutation (IN). Among the IBD patients the only independent factor significantly associated with hyperhomocysteinemia was folate deficiency (p = 0.0002), regardless of the MTHFR or the CBS genotype. CONCLUSIONS: IBD patients have a higher prevalence of hyperhomocysteinemia than do healthy controls. Folate deficiency is the only independent risk factor in developing hyperhomocysteinemia.

Adult↗
PubMed2001

Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

Animals↗
PubMed2001

O-raffinose-polymerised haemoglobin. A biochemical and pharmacological profile of an oxygen carrier.

Haemoglobin-based oxygen carriers (HBOCs) represent an interesting class of blood substitutes which are undergoing advanced clinical trials. The therapeutic goal of these compounds is to avoid or reduce blood transfusion in different surgical and medical situations of acute haemoglobin deficiency. Their main advantages include availability in large volumes, storage for prolonged periods, rapid administration (without typing and cross matching) and sterilisation by pasteurisation. Their main known disadvantages are reduced circulation half-life, haemodynamic and gastrointestinal perturbations, probably related to nitric oxide (NO) scavenging, free radical induction, and alterations of biochemical and haematological parameters (increases in liver enzymes levels, platelet aggregation). Cell-free o-raffinose cross-linked and oligomerised human haemoglobin (O-r-poly-Hb) (Hemolink, Hemosol, Canada) is a modified haemoglobin with molecular weight ranging from 32- > 500 kDa. Its affinity for oxygen appears lower than normal blood and an n (Hill coefficient) value of about 1 indicates a very low degree of co-operativity. Probably related to the low O2 affinity value and to the high molecular weight, O-r-poly-Hb has been shown to induce lesser haemodynamic perturbations than other first generation modified haemoglobins. This HBOC is in Phase III clinical trials in cardiac and orthopaedic surgery for perioperative haemodilution, at doses from 25 g (250 ml)-100 g (1000 ml).

Animals↗
PubMed2000

Anomalous dystroglycan in carcinoma cell lines.

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected beta-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous approximately 31 kDa beta-dystroglycan band. alpha-Dystroglycan was udetectable in most of the cell lines in which beta-dystroglycan was found as a approximately 31 kDa species. An anomalous approximately 31 kDa beta-dystroglycan band was also observed in N-methyl-N-nitrosurea-induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low- and high-ionic strength, we demonstrated that both the 43 kDa and approximately 31 kDa beta-dystroglycan bands harbour their transmembrane segment.

Animals↗
PubMed2000

Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.

The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.

Amino Acid Sequence↗
PubMed2000

Large, sustained cardiac lipid peroxidation and reduced antioxidant capacity in the coronary circulation after brief episodes of myocardial ischemia.

OBJECTIVES: We sought to investigate whether a brief episode of myocardial ischemia produces a detectable cardiac oxidative stress in patients undergoing elective coronary angioplasty (PTCA). BACKGROUND: Although cardiac oxidative stress has been clearly demonstrated in experimental models of ischemia-reperfusion, its presence in patients after transient myocardial ischemia is still unclear. METHODS: In order to evaluate oxidative stress in ischemic cardiac regions, plasma conjugated dienes (CD), lipid hydroperoxides (ROOHs) and total antioxidant capacity (TRAP), independent indexes of oxidative stress, were measured in the aorta and great cardiac vein (GCV) before (t0), 1, (t1), 5 (t5) and 15 min (t15) after first balloon inflation in 15 patients undergoing PTCA on left anterior descending coronary artery (Group 1); six patients with right coronary artery stenosis (Group 2), which is not drained by the GCV, were studied as controls. RESULTS: In Group 1 at baseline, CD and ROOHs levels were higher in GCV than in aorta (p < 0.01 for both), and TRAP levels were lower (p < 0.01). Aortic levels of CD, ROOHs and TRAP did not change at any time after to; venous levels of CD and ROOHs levels markedly increased at t1, at t5 and remained elevated at t15 (p < 0.01 for all comparisons vs. to); venous levels of TRAP decreased at t1 and t5 (p < 0.01 vs. t0) and returned to normal at t15. In Group 2, CD, ROOHs and TRAP levels were similar in the aorta and GCV and did not change throughout the study. CONCLUSIONS: Short episodes of myocardial ischemia during PTCA induce a sustained oxidative stress, which is detectable in the venous effluent of reperfused myocardium.

Angioplasty, Balloon, Coronary↗
PubMed2000

Adult and fetal haemoglobin J-Sardegna [alpha50(CE8)His-->Asp]: functional and molecular modelling studies.

Haemoglobin (Hb) J-Sardegna [alpha50(CE8)His-->Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 degrees C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 degrees C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the alpha(1)-beta(1) interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [alpha50(CE8)His-->Arg] at the same position.

2,3-Diphosphoglycerate↗