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Biomedical subjects

E Meucci

Publications and source records attributed to E Meucci.

At least 19 recordsLinked to original sources
PubMed1992

Conformational stability of bovine alpha-crystallin. Evidence for a destabilizing effect of ascorbate.

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.

Acrylamide↗
PubMed1992

Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1.

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.

Alanine Transaminase↗
PubMed1991

Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging.

Mild oxidative stress, as elicited by ascorbate, oxygen, and trace metals, affects the binding properties of human serum albumin via purely conformational changes. In fact, no gross alteration can be observed in the electrophoretic and chromatographic patterns of albumin, whereas localized modifications are indicated by the changes in absorption and fluorescence spectra and in polarization degree. The oxidized protein presents a small increase of bityrosine production and a time-dependent increase in the content of carbonyl groups, whereas proteolytic susceptibility is unchanged. A higher affinity for cis-parinaric acid and a slight loss of solubility in high salt indicate a greater surface hydrophobicity. Pinpoint denaturation of the albumin molecule is also suggested by a decreased "esterase" activity in the presence of p-nitrophenyl acetate. Conformational stability evaluated through thermal shock and addition of moderate amounts of guanidine indicate that the oxidized protein is more heat-resistant, less flexible, and more rigid than the native one. Although limited, structural damages afforded by the oxidative stress cause alterations of albumin binding properties as documented by experiments with probes and physiological ligands. The loss of biological activity of human serum albumin induced by ascorbate system appears of medical relevance, because it can affect drug metabolism and particularly drug tolerance in the elderly.

Ascorbic Acid↗
PubMed1990

Prooxidant action of desferrioxamine: enhancement of alkaline phosphatase inactivation by interaction with ascorbate system.

Desferrioxamine (DFO) nearly doubles alkaline phosphatase oxidative inactivation by the ascorbate system. The effect is dependent on ascorbate and desferrioxamine concentrations, exhibiting in both cases a saturation mechanism. Conversion of desferrioxamine to ferrioxamine abolishes the prooxidant action. Desferrioxamine also increases ascorbate-dependent oxygen consumption and nitroblue tetrazolium reduction. Superoxide dismutase, which blocks the desferrioxamine enhancing effect on enzyme inactivation, markedly slows down nitroblue tetrazolium reduction as well as oxygen consumption by ascorbate plus desferrioxamine, while it fails to protect against the ascorbate system alone. Therefore, in the presence of desferrioxamine, the metal-catalyzed ascorbate autooxidation becomes superoxide-dependent and thus inhibitable by superoxide dismutase. Catalase, peroxidase, and ascorbate oxidase protect alkaline phosphatase from inactivation by both ascorbate and ascorbate-desferrioxamine systems. Hemin shields the enzyme from ascorbate plus DFO attack but not from ascorbate alone. In air-saturated solution, desferrioxamine seems to mediate one electron transfer from ascorbate to oxygen, generating superoxide anions, which can either trigger a Fenton reaction or produce desferal nitroxide radicals. In the absence of oxygen, ascorbate alone is ineffective, but the ascorbate plus desferrioxamine system still inactivates the enzyme; catalase, peroxidase, and ascorbate oxidase, but not superoxide dismutase, afford protection.

Alkaline Phosphatase↗
PubMed1989

Binding in vivo of selective mu and delta opioid receptor agonists: opioid receptor occupancy by endogenous enkephalins.

The in vivo binding properties of cerebral mu and delta opioid receptors were investigated in mice after the intrastriatal injection of [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or [3H][D-Thr2,Leu5]enkephalyl-Thr (DTLET). Both peptides exhibited similar diffusion kinetics in the brain and 30-40% of [3H]DAGO or [3H]DTLET was shown to be present in the tissue 15 min after injection when maximal binding was observed. The specific binding of both agonists, defined as the fraction of the radioactivity bound to brain membranes which was displaced by 10 nmol of cold ligand, was reversible, saturable and displayed a pharmacological profile similar to that found in in vitro experiments. At doses producing a similar analgesic effect in the hot-plate test in mice, DTLET occupied 64% of delta sites and DAGO 15% of mu sites. However, because of the residual cross-reactivity of DTLET for mu sites, it appeared that both ligands occupied a similar number of mu receptors at their ED50 values, thus supporting a preferential involvement of mu opioid binding sites in the supraspinal pain control. [Met5]enkephalin inhibited the in vivo binding of both agonists only when the peptide was protected from degradation by the co-administration of a mixed inhibitor of enkephalin degrading enzymes RB38A (N[3(R)(hydroxyaminocarbonyl)-2-benzyl-1-oxopropyl]- L-phenylalanine). Unlike thiorphan, 5 nmol RB38A alone was able to inhibit [3H]DAGO binding by 60%. This result is the first direct demonstration of the existence of an in vivo tonic control of mu opioid receptor occupation by endogenous opioid peptides.

Animals↗
PubMed1989

Carcinoma following gastric surgery: a national survey.

Definitive results of a national survey on the incidence of carcinoma following gastric surgery are reported. Data concern 8427 cases of surgically treated gastric cancer. 558 were affected by carcinoma following gastric surgery and 471 of them were operated on, representing 5.59% of all gastric cancers (8427) submitted to surgery. The statistical analysis of results has pointed out the need for a close follow-up program in patients over 50 years, operated on for benign gastroduodenal disease, especially 10 years postoperatively and in case of pre-cancerous lesions.

Aged↗
PubMed1988

Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.

No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.

Alkaline Phosphatase↗
PubMed1987

Alkaline phosphatase inactivation by mixed function oxidation systems.

Alkaline phosphatase is inactivated by mixed function oxidation systems. OH. radicals, generated via an ascorbate-modified Haber-Weiss cycle or a Fenton-type reaction, seem to be responsible for the protein oxidative damage. Experiments with hydroxyl radical scavengers, enzyme substrates, products, and metal cofactors suggest that a "site-specific" radical attack takes place at or near the active center. Vitamin E fails to protect alkaline phosphatase; uric acid, instead, is particularly effective in shielding the protein against covalent modifications.

Alkaline Phosphatase↗
PubMed1986

The relationship between the optical properties and the kinetic behaviour of ascorbate-inhibited alkaline phosphatase.

Aromatic residues of bovine kidney alkaline phosphatase appear to be involved in the interaction with ascorbate, as shown by the strong quenching of intrinsic fluorescence and absorption. Difference u.v.-absorption spectra clearly indicate that conformational changes also occur. The pH value at which the greatest fluorescence deactivation is found is close to that necessary for optimal catalytic activity and for maximal inhibition by ascorbate. A protective effect against ascorbate is afforded by Pi. Time profiles of inactivation on one side and of absorbance and emission quenching on the other display opposite behaviours. Attempts to reverse the effects by the use of KOH fail to restore enzyme activity or to modify the spectral effects of ascorbate. The protein alterations are related, directly or indirectly, to the enzyme active centre and can be probably ascribed to the redox and chelating properties of ascorbate.

Alkaline Phosphatase↗
PubMed1985

Ascorbic acid stability in aqueous solutions.

Different water purity provokes a great variation of the stability of ascorbic acid and isoascorbic acid solutions. The effect of temperature on ascorbate aerobic oxidation was assessed by means of Arrhenius plots from which thermodynamic parameters were derived. The presence of bovine serum albumin drastically reduces the vitamin oxidation rate regardless of stereoisomerism. On the other hand the interaction with alkaline phosphatase, an enzyme inhibited by preincubation with vitamin C, does not modify significantly the stability in the experimental conditions used.

Alkaline Phosphatase↗
PubMed1984

Characterization of alkaline phosphatase inactivation by ascorbic acid.

Ascorbic acid, isoascorbic acid and dehydroascorbic acid inhibit bovine kidney alkaline phosphatase activity. Ascorbic acid free radicals seem not to be involved. Dialysis does not make the inactivation reversible. A competitive mechanism can be inferred from experiments with phosphate and substrates, which block the activity decay. The influence of temperature, pH, other inhibitors and tertiary structure modifications on the inactivation process is also investigated.

Alkaline Phosphatase↗
PubMed1984

Studies on the selective chemical inhibition by urea of alkaline phosphatase isoenzymes in the reaction course.

Urea inhibits the activity of alkaline phosphatase during the reaction course. The inactivation is progressively stronger for the placental, intestinal and renal subforms. Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin and enzyme concentration on the inactivation mechanism is evaluated. In all experimental conditions the process follows pseudofirst-order kinetics and the inactivation profiles are distinct and typical for each enzymatic subform. With a simple graphical analysis, a single inactivation curve in controlled experimental conditions, allows the identification of each isoenzyme from the slope and the calculation of the respective fractional amount from the intercept of the time-activity plot.

Alkaline Phosphatase↗