Alessandro Finazzi Agrò

The biochemical mechanism of selective heat sensitivity of cancer cells: I. Studies on cellular respiration☆

Resume On a etudie leffet de la temperature, entre 38 et 43° C, sur lincorporation des precurseurs marques dans le DNA, RNA et les proteines des cellules de lhepatome de Novikoff et de lhepatome 5123 de Morris. La temperature a provoque une inhibition marquee; celle-ci a ete observee dans les deux types de cellules cancereuses, alors que les cellules de foie regenerant ne sont pas affectees. Cet effet de la temperature a ete observe aussi dans les cellules dun osteosarcome humain. Les preparations acellulaires sont insensibles a la temperature. Le d-glucose, ajoute au melange dincubation, cause une inhibition tres evidente. La filipine et lethanol inhibent lincorporation dans les cellules cancereuses a des concentrations qui nont pas deffet appreciable sur les cellules du foie regenerant. Pour expliquer le mecanisme daction de la chaleur sur les cellules cancereuses, on peut envisager des alterations des membranes.

The biochemical mechanism of selective heat sensitivity of cancer cells: II. Studies on nucleic acids and protein synthesis☆

Resume On a etudie leffet de la temperature, entre 38 et 43°C, sur lincorporation des precurseurs marques dans le DNA, RNA et les proteines des cellules de lhepatome de Novikoff et de lhepatome 5123 de Morris. La temperature a provoque une inhibition marquee; celle-ci a ete observee dans les deux types de cellules cancereuses, alors que les cellules de foie regenerant ne sont pas affectees. Cet effet de la temperature a ete observe aussi dans les cellules dun osteosarcome humain. Les preparations acellulaires sont insensibles a la temperature. Le d -glucose, ajoute au melange dincubation, cause une inhibition tres evidente. La filipine et lethanol inhibent lincorporation dans les cellules cancereuses a des concentrations qui nont pas deffet appreciable sur les cellules du foie regenerant. Pour expliquer le mecanisme daction de la chaleur sur les cellules cancereuses, on peut envisager des alterations des membranes.

Enzyme defense against reactive oxygen derivatives. II. Erythrocytes and tumor cells

SummaryThe enzymatic destruction of oxidizing products produced during metabolic reduction of oxygen in the cell (such as singlet oxygen, H2O2 and OH radical) involves the concerted action of superoxide dismutase-which removes O2- and yields H2O2-and H2O2 removing enzymes such as catalase and glutathione peroxidase. A difference in distribution or ratio of these enzymes in various tissues may result in a different reactivity of oxygen radicals.It was found that in red blood cells superoxide dismutase and catalase are extracted in the same fraction as hemoglobin, while glutathione peroxidase appears to be “loosely” bound to the cellular structure. This suggests that in red blood cells catalase acts in series with superoxide dismutase against bursts of oxygen radicals formed from oxyhemoglobin, while glutathione & peroxidase may protect the cell membrane against low concentrations of H2O2. On the other hand, catalase activity is absent in various types of ascites tumor cells, while glutathione peroxidase and superoxide dismutase are found in the cytoplasm. However, the peroxidase/dismutase ratio is lower than in liver cells, and this may provide an explanation for the higher susceptibility of tumor cells to treatments likely to involve oxygen radicals.

Structure and Function of Amine Oxidases

Amine oxidases are enzymes widely distributed among living organisms. They catalyze the oxidative deamination of many biologically important amines with the formation of the corresponding aldehyde, hydrogen peroxide and ammonia according to the equation RCH2NH2 + O2 + H2O → RCHO + H2O2 + NH3

A possible role for rhodanese: The formation of ‘labile’ sulfur from thiosulfate

Rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) is a well-known mitochondrial protein [ 1,2] . In spite of its widespread occurrence and abundance, its physiological role is very uncertain. In vitro it catalyzes the transport of sulfur from thiosulfate to a nucleophilic acceptor (cyanide, reduced lipoate) [3,4] by a double displacement reaction with the formation of an intermediate sulfur-enzyme complex [5,6] . Although some cyanide may be formed in vivo, this seems insufficient to explain the ubiquity and abundance of rhodanese. The possible significance of its action on reduced lipoate is also unclear. In the present paper a new role for rhodanese is outlined, namely the possibility that the enzyme may contribute to the formation of ‘labile sulfur’ in non-heme iron proteins from thiosulfate.

Epigenetic mechanisms and endocannabinoid signalling

The endocannabinoid system, composed of endogenous lipids, their target receptors and metabolic enzymes, has been implicated in multiple biological functions in health and disease, both in the central nervous system and in peripheral organs. Despite the exponential growth of experimental evidence on the key role of endocannabinoid signalling in basic cellular processes, and on its potential exploitation for therapeutic interventions, much remains to be clarified about the respective regulatory mechanisms. Epigenetics refers to a set of post‐translational modifications that regulate gene expression without causing variation in DNA sequence, endowed with a major impact on signal transduction pathways. The epigenetic machinery includes DNA methylation, histone modifications, nucleosome positioning and non‐coding RNAs. Due to the reversibility of epigenetic changes, an emerging field of interest is the possibility of an ‘epigenetic therapy’ that could possibly be applied also to endocannabinoids. Here, we review current knowledge of epigenetic regulation of endocannabinoid system components under both physiological and pathological conditions, as well as the epigenetic changes induced by endocannabinoid signalling.

Luminescence of the copper—carbon monoxide complex of Neurospora tyrosinase

Spectroscopic studies of tyrosinase and hemocyanin have indicated that the copper-sites of both proteins are very similar. Thus the stoichiometry of oxygen binding [ I2], EPR meas~lre~~~ents on various derivatives [3,4], magnetic susceptibility measurements [5.6] and resonance Raman spectroscopy [7] suggest the presence of a pair of antiferromagnetically coupled copper-ions separated by 36 8. Moreover hernocyanin and tyrosinase both bind carbon monoxide [8,9]. We have reported that the carbon monoxide complex of hemocyanins is luminescent [ 10.1 11. Upon excitation in the ultraviolet region, a strong emission band is observed above 500 nm. Excitation and difference absorption spectra, as well as CO titrations, clearly indicate that the emission originates from the copper-CO chromophore. In view of the above similarities between hemocyanin and tyrosinase, we have studied the binding of CO to ~e~~r~sp~~~ tyrosinase by fluorescence spectroscopy, and observed that the luminescence characteristics of CO-tyrosinase are similar to those reported for CO-hemocyanins [IO,1 I]. This result emphasizes the close structural relationships existing between the active site of these two classes of proteins, notwithstandmg the overall functional differences; moreover it confirms the potentialities of fluorescence spectroscopy of the CO complexes of these proteins, to investigate the structure of their binuclear copper sites. 2. Materials and methods

Reaction of carbon monoxide with hemocyanin: Stereochemical effects of a non-bridging ligand

Abstract The carbon monoxide binding equilibria and kinetics of a number of molluscan and arthropodal hemocyanins have been investigated employing the visible luminescence of the carbon monoxide-copper complex. Proteins from both phyla, in oligomeric and monomeric form, bind carbon monoxide non-co-operatively; the reaction is largely enthalpy driven is associated with a small unfavourable entropy change. Molluscan hemocyanins display a carbon monoxide affinity (p50 = 1 to 10mm Hg) higher than that of arthropodal hemocyanins (p50 = 100 to 700mm Hg), and only Panulirus interruptus hemocyanin, among those studied here, exhibits a small Bohr effect. The observed differences in equilibrium constant are kinetically reflected in differences in the carbon monoxide dissociation rate constant, which ranges from 20 to 70 s−1 for molluscan hemocyanins and from 200 to 9000 s−1 for arthropodal hemocyanins; on the other hand the differences in the combination rate constants between the two phyla are considerably smaller. A comparison of the equilibrium and kinetic results shows some discrepancies between the two sets of data, suggesting that carbon monoxide binding may be governed by a complex mechanism. The correlation between the ligand binding properties and the stereochemistry of the active site is discussed in the light of the knowledge that, while oxygen is bound to both copper atoms in a site, carbon monoxide is a “non-bridging” ligand, being bound to only one of the metals.

Activity and expression of diamine oxidase in lentil seedling under different growth conditions

The activity and expression of diamine oxidase during the germination of lentil seeds and in the course of anoxic and thermal stress have been studied. Diamine oxidase activity, as well as the seedlings growth rate, was found to be markedly higher in dark-grown lentil seedlings than in the light-grown ones. The same was true for the respective protein and mRNA amounts. The specific activity of diamine oxidase was decreased by anoxic stress and not affected by thermal stress. The possible physiological meaning of these findings is discussed.

The effect of azide on the spectral and catalytic properties of ascorbate oxidase

Summary(1)45% of the total copper of green zucchini ascorbate oxidase is EPR-detectable. At least two species of copper are present, one with a small A∥ (Type 1) and one with a large A∥ (Type 2). Computer simulated spectra indicated 50% contribution by each type of copper.(2)Azide inhibited ascorbate oxidase activity by an uncompetitive mechanism. EPR and optical spectra performed on titration of ascorbate oxidase with azide indicated the formation of a copper-azide complex. The Type 2 copper appears to be the binding site of azide. The involvement of the EPR non-detectable copper as an anion binding site with high affinity toward azide can not be excluded.