Carlo Crifò

The biochemical mechanism of selective heat sensitivity of cancer cells—IV. Inhibition of RNA synthesis

Abstract The mechanism by which exposure of ascites tumour cells to supranormal temperatures causes an irreversible inhibition of uridine incorporation into RNA was investigated. Heat-treated cells were still able to incorporate labeled nucleotides, and even nucleosides, into RNA, if these precursors were added at sufficiently high concentrations. The passive permeability of the cell membrane increased exponentially with temperature, but this increase was fully reversible. At variance with the results obtained with agents which increase cell permeability, or which inhibit nucleoside permeation, heat treatment of Ehrlich ascites cells did not modify the ability of labeled uridine to be metabolized by these cells. The inhibition of uridine incorporation following heat treatment cannot therefore be attributed to a damage at the cell membrane level; the experimental data can be tentatively accounted for by the hypothesis of a block in the maturation of pre-rRNA into rRNA, with reutilization of the pre-rRNA degradation products.

High sensitivity of plasma membrane ion transport ATPases from human neutrophils towards 4-hydroxy-2,3-trans-nonenal.

Lipid peroxidation results in release of 4-hydroxy-2,3-trans-nonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. The effect of HNE on the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and calmodulin-stimulated Ca(2+)-ATPase has been studied both in erythrocyte ghosts and in neutrophil membrane preparations. Neutrophil Ca(2+)-ATPase was strongly inhibited by micromolar concentrations of HNE (IC(50) = 12 microM), that means in the range of pathophysiologically relevant HNE levels. The IC(50) value for neutrophil Na(+)/K(+)-ATPase was about 40 microM. HNE was considerably less effective against neutrophil Mg(2+)-ATPase and the erythrocyte ghost enzymes (IC(50) values range from 91 to 240 microM). The data suggest that HNE may play a specific role in the regulation of neutrophil calcium homeostasis in response to oxidative stress.

VARIATIONS IN CIRCULAR DICHROISM AND PROTON-NMR RELAXATION PROPERTIES OF MELITTIN UPON INTERACTION WITH PHOSPHOLIPIDS

Melittin, a 26 amino acid cationic peptide extracted from bee venom [ 11, is well known for its ability to interact with phospholipid structures, dramatically increasing the permeability to aqueous solutes of either natural or artificial membranes [2]. Upon interaction with melittin, the aliphatic chains of the phospholipid molecules seem to undergo a pronounced immobilization, as evidenced by infrared and spin label studies [3,4]. As for the melittin molecule, its only tryptophan residue moves, upon interaction with phospholipids, from an aqueous environment to a hydrophobic one, as judged from the blue shift of its fluorescence maximum [5,6]; the changes in fluorescence characteristics are however dependent on the phospholipid used [6]. No change in the infrared absorption or infrared dichroism spectra of the peptide has been evidenced 141. We describe here how the intrinsic circular dichroism of melittin and the NMR relaxation properties of its tryptophan protons are modified upon interaction with phospholipids.

Inhibition of NADPH Oxidase-Mediated Superoxide Radical Formation in PMA-Stimulated Human Neutrophils by 4-Hydroxynonenal- Binding to -SH and -NH2 Groups'

4-Hydroxynonenal (HNE), a major lipid peroxidation product, effectively inhibits the superoxide radical formation by NADPH oxidase of phorbol myristate acetate (PMA)--stimulated human PMNL. The I50 value for the inhibition of NADPH oxidase-mediated superoxide radical formation by 4-hydroxynonenal was found to be 19 microM. The HNE inhibition involves the reaction with both -SH and -NH2 groups. Superoxide formation as final result of the NADPH oxidase cascade was almost completely restored by addition of dithiothreitol. In presence of hydroxylamine only a minor restoration of superoxide radical formation was found. A combination of dithiothreitol and hydroxylamine yielded the greatest recovery. Two other aldehydes with the same chain length as HNE but different binding to lysine, histidine and cysteine residues, trans-2,3-nonenal and nonanal, gave I50 values for the inhibition of NADPH oxidase-mediated superoxide formation rate of 110 microM or > 300 microM, respectively.

The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: I. A Fluorometric and Spectrophotometric Study

The increase of fluorescence emission of lucensomycin occurring upon interaction with cholesterol, either as colloidal suspension or included in phospholipid micelles or in erythrocyte membranes, was described. Colloidal cholesterol differed from that contained in membranes by the kinetic behavior of its interaction with lucensomycin, the reaction being very slow in the former case, very fast in the latter one. Variations of optical density also occurred, though neither the kinetics nor the titration curves were superimposable on those obtained fluorometrically. The stoichiometry was, however, the same.

Adenylosuccinase deficiency: a patient with impaired erythrocyte activity and anomalous response to intravenous fructose

SummaryClinical and biochemical data are presented on an Italian patient with adenylosuccinase deficiency of both erythrocytes and mixed peripheral blood lymphocytes. The erythrocyte enzyme showed normal substrate affinity, but decreased thermal stability. The patient displayed an anomalous response to an intravenous fructose tolerance test with a rise in plasma [Mg2+] and [K+] and a drop in plasma levels of inorganic phosphate, glucose, urate and succinylnucleosides upon fructose injection.

β-carotene degradation products - formation, toxicity and prevention of toxicity.

Carotenoids are widely used as important micronutrients in food. Furthermore, carotenoid supplementation has been used in the treatment of diseases associated with oxidative stress such as various types of cancer, inflammatory diseases or cystic fibrosis. However, in some clinical studies harmful effects have been observed, e.g. a higher incidence of lung cancer in individuals exposed to extraordinary oxidative stress. The causal mechanisms of harmful effects are still unclear. Carotenoid breakdown products (CBPs) including highly reactive aldehydes and epoxides are formed during oxidative attacks in the course of antioxidative action. We investigated the formation of CBPs by stimulated neutrophils (and at further conditions), tested the hypothesis that CBPs may exert mitochondriotoxicity and tried to prevent toxicity in the presence of members of the antioxidative network. Stimulated neutrophils are able to degrade beta-carotene and to generate a number of CBPs. Concerning mitochondriotoxicity, we found that CBPs strongly inhibit state 3 respiration of rat liver mitochondria at concentrations between 0.5 and 20 microM. This was true for retinal, beta-ionone, and for mixtures of cleavage/breakdown products. The inhibition of mitochondrial respiration was accompanied by a reduction in protein sulfhydryl content, decreasing GSH levels and redox state, and elevated accumulation of malondialdehyde. Changes in mitochondrial membrane potential favor functional deterioration in the adenine nucleotide translocator as a sensitive target. The presence of additional antioxidants such as alpha-tocopherol, ascorbic acid, N-acetyl-cysteine or others could mitigate mitochondriotoxicity. The findings reflect a basic mechanism of increasing the risk of cancer induced by carotenoid degradation products.

PHOTOHEMOLYSIS OF ERYTHROCYTES ENRICHED WITH SUPEROXIDE DISMUTASE, CATALASE AND GLUTATHIONE PEROXIDASE

Abstract— Cationic liposomes have been used to introduce into human erythrocytes variable amounts of the enzymes superoxide dismutase, catalase and glutathione peroxidase. The behaviour of the enzyme‐enriched erythrocytes toward photohemolysis sensitized by protoporphyrin IX has been studied in comparison to that of erythrocytes treated with empty liposomes. The erythrocytes with increased catalase and glutathione peroxidase activity were more resistant to photohemolysis. In contrast, the erythrocytes enriched with superoxide dismutase hemolyzed faster. The association of two enzymes at a time was also studied

Carotenoid cleavage products modify respiratory burst and induce apoptosis of human neutrophils.

Carotenoid supplementation in the treatment of diseases associated with oxidative stress has been recently questioned because of the cell damage and the increased risk of lung cancer in male smokers. Because of the complex role of neutrophils in lung diseases, we investigated whether carotenoid derivatives could affect respiratory burst and apoptosis of human neutrophils purified from peripheral blood. Stimulation of superoxide production was induced by nanomolar and micromolar concentrations of carotenoid cleavage products with aliphatic chains of different length, but not by carotenoids lacking the carbonyl moiety. The stimulatory effect of carotenoid cleavage products was observed in cells activated by phorbol myristate acetate (PMA), while a slight inhibition of superoxide production was noticed with cells activated by the chemotactic tripeptide N-formyl-Met-Leu-Phe (f-MLP). At higher concentrations, carotenoid cleavage products inhibited superoxide production in the presence of both PMA and f-MLP. In the presence of 20 microM carotenoid cleavage products, inhibition of superoxide production was accompanied by DNA fragmentation and increased level of intracellular caspase-3 activity.

Determination of urinary orotic acid and uracil by capillary zone electrophoresis.

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperomithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.