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Biomedical subjects

B Giardina

Publications and source records attributed to B Giardina.

At least 55 records · Page 3Linked to original sources
PubMed2000

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis.

A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.

Electrophoresis, Capillary↗
PubMed2000

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.

AMP Deaminase↗
PubMed2000

Structural and functional analysis of the two haemoglobins of the antarctic seabird Catharacta maccormicki characterization of an additional phosphate binding site by molecular modelling.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1alpha, G6alpha and HC3alpha, is located between the two alpha chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two beta chains by means of a site-site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.

Adaptation, Physiological↗
PubMed2000

Blood bank conditions and RBCs: the progressive loss of metabolic modulation.

BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD-saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37 degrees C with pyruvate-inosine-phosphate-adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD-saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3.

Adenine↗
PubMed2000

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity.

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)+, only Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 microM) or H2O2 (concentration range 50-1000 microM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)+, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)+/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)+/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)+ production of Fe2+-H2O2-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where "conventional" oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.

Adenosine Diphosphate Ribose↗
PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗
PubMed2000

Myocardial ischemia-reperfusion damage after pacing-induced tachycardia in patients with cardiac syndrome X.

The presence of myocardial ischemia in syndrome X (chest pain, "ischemia-like" electrocardiogram changes, and normal coronary angiograms) is uncertain possibly because, when focally distributed, it may not cause contractile dysfunction or lactate production. We measured lipid hydroperoxides (ROOHs) and conjugated dienes (CDs), two sensitive, independent markers of ischemia-reperfusion oxidative stress, in paired aortic and great cardiac vein blood samples before and after pacing-induced tachycardia in nine patients with syndrome X. Diagnostic ischemic S-T segment changes during pacing were followed by a consistent increase in ROOH and CD levels in the great cardiac vein (from 4.83 +/- 1.18 micromol/l at baseline to 7.88 +/- 1.12 micromol/l and from 0.038 +/- 0.002 to 0.051 +/- 0.003 arbitrary units, respectively, P < 0.01). In controls, ROOH and CD levels did not change after pacing. The large postpacing cardiac release of lipid peroxidation products, consistently observed in all patients and similar to that previously observed after ischemia caused by percutaneous transluminal coronary angioplasty, is consistent with an ischemic origin of syndrome X.

Adult↗
PubMed2000

A new, electrophoretically silent, fetal hemoglobin variant: Hb F-Calabria [Ggamma118(GH1)Phe-->Leu].

Hb F-Calabria [Ggamma118(GH1)Phe-->Leu] is a new fetal hemoglobin variant that was found during routine screening for abnormal hemoglobins in a newborn of Calabrian (Southern Italy) ancestry. The variant chain was identified (acid urea gel electrophoresis of dissociated globin chains in the presence of Triton X-100, and by reversed phase high performance liquid chromatography) as a slightly hydrophilic Ggamma chain. Sequencing of the polymerase chain reaction-amplified exon 3 of the Ggamma-globin gene demonstrated the TTC-->CTC mutation at codon 118 leading to the Phe-->Leu conservative substitution at position GH1. A molecular modeling study supports that the variant might not have clinical implications. This is the 40th example of a Ggamma chain variant.

Amino Acid Substitution↗
PubMed2000

Oxidative stress in ischemia-reperfusion injury: assessment by three independent biochemical markers.

BACKGROUND: Oxidative stress plays a key role in ischemia-reperfusion injury, causing peroxidation of tissue lipids and proteins. However, it is debated whether brief ischemic episodes are sufficient to cause detectable oxidative stress in humans, since biochemical markers used so far in the setting of percutaneous transluminal coronary angioplasty (PTCA) gave conflicting results. METHODS: We determined lipid hydroperoxides (ROOHs), conjugated dienes (CD) and total radical-trapping antioxidant capacity (TRAP), three different independent markers of oxidative stress, in aortic and great cardiac vein blood of 5 patients undergoing PTCA before a single balloon inflation lasting 115 +/- 38 s (t0), and 1 min (t1), 5 min (t5), 15 min (t15) after balloon deflation (Group 1). ROOHs and CD were also determined in aortic and great cardiac vein blood of 5 patients with mitral valve disease (Group 2). RESULTS: In Group 1, great cardiac vein levels of ROOHs and CD at t1 increased by 219% and 79%, respectively, compared to t0 (p < 0.01); this sharp and consistent increase persisted up to t15 (+189% and +63%, respectively, compared to t0; p < 0.01). Great cardiac vein levels of TRAP were significantly lower than aortic levels at t0, and exhibited a further decrease at tl. No significant differences in aortic and great cardiac vein levels of ROOHs and CD at t0 were observed between Group 1 and Group 2. CONCLUSIONS: The three methods we used showed a remarkable sensitivity for the detection of post-ischemic reperfusion injury in cardiac venous blood and may be useful for detecting small foci of ischemia-reperfusion injury in microvascular angina.

Angioplasty, Balloon, Coronary↗
PubMed1999

Structural and functional analysis of the N-terminal extracellular region of beta-dystroglycan.

A protein fragment corresponding to the mouse beta-dystroglycan N-terminal extracellular region from position 654 to 750, beta-DG(654-750) was recombinantly expressed in BL21(DE3) Escherichia coli cells. Secondary structure prediction of the protein fragment reveals about 70% of random coil, as confirmed by circular dichroism analysis. Moreover, fluorescence analysis shows that the tryptophan residue in position 659 lays in a solvent-exposed fashion. These data suggest that the beta-DG(654-750) is likely to have a quite flexible structure and to be only partially folded. Interestingly, the protein still retains its biological function since using solid-phase assays we have detected binding of biotinylated beta-DG(654-750) both to native alpha-dystroglycan and to a recombinant fragment which spans the C-terminal region of alpha-dystroglycan.

Amino Acid Sequence↗
PubMed1999

Quantitative determination of the main glucose metabolic fluxes in human erythrocytes by 13C- and 1H-MR spectroscopy.

Information displayed by homonuclear and heteronuclear spin-coupling patterns in 13C- and 1H-MR spectra allowed us to identify the major lactate isotopomers produced either from [1-(13)C]-glucose or from [2-(13)C]-glucose by human erythrocytes. Relative concentrations of detectable isotopomers were determined by integrating the corresponding MR signals. The interpretation of these data in terms of the fractional glucose metabolised through glycolysis and pentose phosphate pathway was performed by a computer simulation of the metabolism that took into account metabolic schemes pertaining to glycolysis and to the F-type of pentose phosphate pathway. The simulation was organised in a way to anticipate the populations of the isotopomers produced from any precursor at a priori established metabolic steady state. By the simulation, isotopomer populations were determined according to different values of pentose cycle, defined as the flux of glyceraldehyde 3-phosphate originating from pentose phosphate pathway at unitary glucose uptake. The populations of the isotopomers originating from [2-(13)C]-glucose were described by polynomials, and ratios between the polynomials were used in conjunction with 13C- and 1H-MR data to determine pentose cycle values. The knowledge of glucose uptake and of pentose cycle value allowed us to perform accurate measurement of the pentose phosphate pathway flux, of the hexokinase and phosphofructokinase fluxes as well as, indirectly, of the carbon dioxide production.

Erythrocytes↗
PubMed1999

Brief communication: Resistance to Falciparum malaria in alpha-thalassemia, oxidative stress, and hemoglobin oxidation.

A recent survey conducted on Vanuatu Island suggests that resistance to Plasmodium falciparum in alpha-thalassemic individuals may have an immunological basis. This study is important since it seems to undermine the current idea that red-cell genetic defects give protection against falciparum malaria by reducing intraerythrocytic growth and development of the parasite. However, the mechanisms underlying these clinical and genetic observations are not yet fully understood. Based on a review of the relevant literature, we first show that the model based on the interaction between hemoglobin (Hb) and membrane components may provide a molecular basis for the involvement of the immune response in genetic adaptation to malaria. Second, we discuss the main evolutionary implications of the model. Finally, we suggest two approaches by which anthropological studies could provide a useful way of testing the model: 1) analysis of the interactions of malaria-resistance genes with genetic polymorphisms which affect the erythrocyte redox status and 2) study of the antimalarial effects of natural products (introduced as a part of a diet or for traditional antimalarial therapy) capable of interfering with the Hb/membrane interaction.

Animals↗
PubMed1999

Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers.

Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically significant difference between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were significantly (p < 0.05) higher in smokers. Smoking of a single cigarette induced a significant reduction in glutathione concentration (p < 0.05). Salivary antioxidant power may affect individual sensitivity toward tobacco stress.

Adult↗
PubMed1999

Determination of copper, zinc, and selenium in human plasma and urine samples by potentiometric stripping analysis and constant current stripping analysis.

Potentiometric stripping analysis and constant current stripping analysis are proposed as routine methods for analysis of copper, zinc and selenium in plasma and urine samples. The analytical performance of these methods is comparable with that reported for atomic absorption spectrometry. However the low cost, greater simplicity of the apparatus, and the facility of execution make this methodology a valid candidate for routine application in Clinical Chemistry laboratories.

Copper↗
PubMed1999

Purine metabolites and malondialdehyde in platelets of diabetic patients.

The concentration of some of the purine nucleotides and their metabolites together with that of malondialdehyde (MDA) have been measured in resting and stimulated platelets of type 1 and type 2 diabetic patients. While control platelets show a net decrease of guanosine triphosphate (GTP) (3.1 vs. 2.3 nmol per 10(9) platelets) and guanosine diphosphate (GDP) (3.0 vs. 2.0 nmol per 10(9) platelets) and a significant increase of adenosine (0.04 vs. 0.55 nmol per 10(9) platelets) with platelet stimulation, platelets of type 1 and type 2 diabetic patients have a lesser change of these metabolites (GTP, 2.6 vs. 2.4; GDP, 2.3 vs. 2.4; adenosine, 0.04 vs. 0.30 (P < 0.05 vs. control) nmol per 10(9) platelets in type 1 diabetics; GTP, 2.4 vs. 2.7; GDP, 2.4 vs. 2.1; adenosine, 0.08 vs. 0.32 (P < 0.05 vs. control) nmol per 10(9) platelets in type 2 diabetics). These results indicate that the change (stimulated minus resting) of GTP, GDP and adenosine in diabetic platelets is significantly different from that of controls (P < 0.001). Moreover, the amount of MDA produced during platelet activation seems to be lower than controls only in type 2 diabetes (1.81 vs. 2.86 nmol per 10(9) platelets, P < 0.05). These results seem to indicate that a difference in the pattern of platelet nucleotides could be an important feature even in well-controlled diabetes, while MDA is probably modified only in association with the late vascular complications of diabetes.

Adenosine↗
PubMed1999

The myoglobin of Emperor penguin (Aptenodytes forsteri): amino acid sequence and functional adaptation to extreme conditions.

In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

Adaptation, Physiological↗
PubMed1999

Bezafibrate as differentiating factor of human myeloid leukemia cells.

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.

Acetylcysteine↗