Papers by Alessandro Finazzi-Agró
Biosensors and Bioelectronics, 2003
Previous finding shown that the composition of the breath of patients with lung cancer contains i... more Previous finding shown that the composition of the breath of patients with lung cancer contains information that could be used to detect the disease. These volatiles are mainly alkanes and aromatic compounds. Sensor arrays technology (electronic nose) proved to be useful to screen samples characterised by different headspace composition. Here we investigated the possibility of using an electronic nose to check whether volatile compounds present in expired air may diagnose lung cancer. Breath samples were collected and immediately analysed by an electronic nose. A total of 60 individuals were involved in the experiment. 35 of them were affected by lung cancer, 18 individuals were measured as reference and nine were measured after the surgical therapy. Two individuals were measured twice, before and after the surgical therapy, for a total of 62 measurements. An electronic nose, composed by eight quartz microbalance (QMB) gas sensors, coated with different metalloporphyrins, was used. These sensors show a good sensitivity towards those compounds previously indicated as possible lung cancer markers in breath. The application of a 'partial least squaresdiscriminant analysis' (PLS-DA) found out a 100% of classification of lung cancer affected patients, 94% of reference was correctly classified. The class of post-surgery patients were correctly individuated in 44% of the cases, while the other samples were classified as healthy references. The alteration of breath composition induced by the presence of lung cancer was enough to allow a complete identification of the sample of diseased individuals. Extended studies are necessary to evaluate the resolution of the method, namely the stage at which the disease may be identified in order to use this instrument for early diagnosis.
PubMed, Mar 1, 1968
ABSTRACT The effect of temperature on respiration and anaerobic glycolysis of Novikoff and minima... more ABSTRACT The effect of temperature on respiration and anaerobic glycolysis of Novikoff and minimal deviation 5123 hepatoma was investigated. Exposure of tumor cells at 43°C in aerobic conditions caused an inhibition of oxygen uptake and also, to a minor extent, of anaerobic glycolysis. Regenerating liver cells were not inhibited. Disrupted tumor cells were temperature insensitive. Ethanol caused a severe inhibition of respiration in Novikoff hepatoma cells. The effect of ethanol is increased by the higher temperature. Filipin inhibited oxygen uptake by Novikoff hepatoma. The effect was not enhanced by temperature. Regenerating liver was inhibited only by much higher concentrations of ethanol and filipin. The inhibition by ethanol was also in this case enhanced by a higher temperature. The lability of some organized system (i.e. surface or lysosomal membranes) is suggested to explain the observed behaviour of tumor cells.ZusammenfassungDer Einfluss von Temperatur auf Atmung und anaerobe Glykolyse von Novikoff und Morris 5123 Hepatomen wurde untersucht. Eine Temperatur von 43°C bewirkte in Tumorzellen unter aeroben Bedingungen eine Hemmung der O2 Aufnahme und, zu einem geringeren Grad, auch der anaeroben Glykolyse. Regenerierende Leberzellen wurden nicht gehemmt. Zerstörte Tumorzellen zeigten sich temperaturunempfindlich. Aethanol bewirkte eine starke Hemmung der Atmung in Novikoff-Hepatomzellen. Dises Wirkung von Aethanol nimmt mit höherer Temperature zu. Filipin hemmte die O2-Aufnahme von Novikoff-Hepatomen. Temperatur hatte keinen Einfluss auf die Wirkung. Regenerierende Leberzellen wurden nur durch viel höhere Konzentrationen von Aethanol und Filipin gehemmt. Auch in diesem Fall verstärkte eine Erhöhung der Temperatur die Hemmung durch Aethanol. Zur Erklärung dieses Verhaltens der Tumorzellen wird die Labilität eines organisierten Systems (z.B. Oberflächen oder lysosomale Membranen) in Betracht gezogen.
Plant Physiology, 1998
A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogenei... more A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105m−1cm−1 and 6000 m−1cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived...
IUBMB Life, 1996
A simple model, 4-tert-butyl-l,2-benzoquinone, was chosen to study the hydroxylation step of the ... more A simple model, 4-tert-butyl-l,2-benzoquinone, was chosen to study the hydroxylation step of the tyrosine-derived Dopaquinone residue at the active site of copper amine oxidases in the selfcatalytic generation of the Topaquinone cofactor. This hydroxylation step was studied both in the presence and absence of free copper(II), and was found to be dependent on pH value but not on the presence of metal ions. It is therefore proposed that, hydroxide ion and not water should be the true reactive species in this key biosynthetic step of the Topaquinone cofactor, and that the active site Cu 2÷ is implied, at this point of cofactor biosynthesis, in the quinonisation of Topa rather than in the hydroxylation of Dopaquinone.
FEBS Letters, 1984
The reaction with substrate of plant amine oxidases from either Euphorbia latex or lentil seedlin... more The reaction with substrate of plant amine oxidases from either Euphorbia latex or lentil seedlings in the presence of cyanide led to the appearance of a free-radical type of ESR spectrum with well-resolved hyperline structure, which is very likely due to coupling with various protons over an aromatic ring. It was established that the free-radical spectrum was not derived from the substrate, since either aromatic or aliphatic substrates gave rise to identical spectra. The presence of copper is essential to the appearance of the freeradical ESR signal.
The copper enzyme ascorbate oxidase ( E.C. 1.10.3.3 ) belongs to the group of “blue oxidases”. Ac... more The copper enzyme ascorbate oxidase ( E.C. 1.10.3.3 ) belongs to the group of “blue oxidases”. According to recent investigations of Avigliano et al.(1983) the enzyme has a molecular weight of 145,000 with a copper content of 8/146,000 Mr . The molecule consists of two non-covalently linked subunits of slightly different molecular weight (75,000 and 72,000 respectively) . The spectroscopic properties of ascorbate oxidase indicate that the enzyme contains all three different types of copper atoms (Fee, 1975; Malmstroem, 1982).
Progress in clinical and biological research, 1988
Journal of Biological Chemistry, 2001
Transglutaminases (TGases) are seven enzymes, cross-linking proteins by ␥-glutamil-⑀-lysine bonds... more Transglutaminases (TGases) are seven enzymes, cross-linking proteins by ␥-glutamil-⑀-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and ⌬3 (deletion of exon 3), we identified two new splicing variants, ⌬11 and ⌬3⌬11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, ⌬3, ⌬11, and ⌬3⌬11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and ⌬11 are active, whereas ⌬3 and ⌬3⌬11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitro crosslinking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentin in vitro.
Journal of Fluorescence, 2003
The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, a... more The dipolar relaxation process induced around tryptophan, indole and tyrosine in viscous media, as well as in several single tryptophan-containing proteins (staphylococcal nuclease, ribonuclease T1, melittin and albumin), has been studied by dynamic fluorescence measurements. A new theoretical model has been developed, including the relaxation dynamics directly in the fluorescence decay function. The phase shift and demodulation data have been fitted with this new algorithm which allows to resolve the different relaxation times influencing the fluorophore excited state. These parameters are in a good agreement with those measured with the traditional time-resolved emission spectroscopy. The results indicate that indeed a correlation exists between the radiative rate change obtained with the new model and the temporal spectral shift reported in the literature. Finally, this new approach has also been extended to the case of superoxide dismutase and phosphofructokinase, allowing to measure the relaxation time even in proteins lacking a temporal spectral shift during the fluorphore's lifetime.
Molecular and Cellular Biochemistry, 1976
European Journal of Biochemistry, 1999
The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochl... more The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.
FEBS Letters, 1978
Thus, in vitro studies show that, in the presence of air, the protein catalyses the oxidation of ... more Thus, in vitro studies show that, in the presence of air, the protein catalyses the oxidation of Fe’*, which is then incorporated into the shell. In a previous paper [3] we have shown that in anaerobiosis the protein binds some Fe2+ and con- verts it to Fe3+ on admission of oxygen. In this respect the protein acts as an oxidoreductase; there- fore, it seemed of interest to measure the stoichiom- etry of the reaction between oxygen and iron as catalysed by apoferritin. The results indicate that 4 FeV are oxidized per oxygen molecule and that the product of oxygen reduction is water. The presence of catalase does not affect the time course nor the stoichiometry of the reaction. STOICHIOMETRY OF IRON OXIDATION BY APOFERRITIN
A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogenei... more A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo-and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 ؋ 10 5 M ؊1 cm ؊1 and 6000 M ؊1 cm ؊1 , respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinonecontaining amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone. Copper-amine oxidases (amine oxygen oxidoreductase deaminating, copper containing; EC 1.4.3.6) are widespread enzymes oxidizing primary amines with the formation of the corresponding aldehyde, ammonia, and hydrogen peroxide: R-CH 2-NH 2 ϩO 2 ϩH 2 O3 R-CHOϩNH 3 ϩH 2 O 2. These enzymes are ubiquitous in nature, occurring in microorganisms (fungi and bacteria; Cooper et al., 1992), plants (Medda et al., 1995a), and mammals (McIntire and Hartmann, 1993). The crystal structures of copper-amine oxidases from Escherichia coli (Parson et al., 1995) and pea seedlings (Kumar et al., 1996) were recently determined. Amine oxidases are homodimers of 70-to 95-kD subunits. Each subunit contains a tightly bound Cu(II) center that is essential for the enzyme redox activity (Dooley et al., 1991; Medda et al., 1995b), and TPQ is formed by a posttransla-1 This study was partially supported by Ministero Università Ricerca Scientifica e Technologica funds (60%) and by the European Commission (European Social Funds).
Cancer Research, 1971
The incorporation of thymidine in Novikoff hepatoma cells and Ehrlich ascites cells was severely ... more The incorporation of thymidine in Novikoff hepatoma cells and Ehrlich ascites cells was severely and irreversibly inhibited by the polyenic antibiotics filipin and lucensomycin, known for their action on phospholipid-cholesterol membranes. No inhibition could be shown on ...
Vitamins & Hormones, 2002
Endocannabinoids are a new class of lipid mediators, which includes amides and esters of long-cha... more Endocannabinoids are a new class of lipid mediators, which includes amides and esters of long-chain polyunsaturated fatty acids. Anandamide (I) and 2-arachidonoylglycerol (II) are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Ag-tetrahydrocannabinol (m), the active principle of Cannabis sativa preparations such as hashish and marijuana. The pathways leading to the synthesis and release
Journal of Bioluminescence and Chemiluminescence, 1997
Several lipoxygenase products were able to enhance ultraweak light emission and membrane permeabi... more Several lipoxygenase products were able to enhance ultraweak light emission and membrane permeability of human erythroleukemia K562 cells. In particular, 13-hydroperoxyoctadecadienoic acid (13-HPOD) was more effective than hydrophilic hydroperoxides, like H2O2 and tert-butyl hydroperoxide. The enhancement of luminescence induced by 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was inhibited by superoxide and hydroxyl radical scavengers. The effect of 13-HPOD was potentiated by the calcium ionophore A23187 and inhibited by the calcium chelator EDTA, and was observed also in liposomes containing unsaturated lipids. Cholesterol enrichment, which decreases the membrane fluidity, did not modify the effect of 13-HPOD on K562 cells.
Journal of Molecular Biology, 1981
The carbon monoxide binding equilibria and kinetics of a number of molluscan and arthropodal hemo... more 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-cooperatively ; the reaction is largely enthalpy driven, and is associated with a small unfavourable entropy change. Molluscan hemocyanins display a carbon monoxide affinity (p,, = 1 to 1Omm Hg) higher than that of arthropodal hemocyanins (p,c = 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-i for molluscan hemocyanins and from 200 to 9000 s-' 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.
Archives of Biochemistry and Biophysics, 1975
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Papers by Alessandro Finazzi-Agró