Substrate specificity of copper-containing plant amine oxidases.

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.

Biogenic amines and apoptosis: minireview article.

The programmed cell death is a very complex mechanism involving many factors, among them the intracellular concentration of biogenic amines (BA) appears to be important for apoptosis triggering. The mitochondrial damage is imputable to hydrogen peroxide and aldehydes, produced by amine oxidases (AO)-mediated oxidation of BA. On the other hands, the apoptosis protection observed by high BA concentration appears to be related to their scavenger effect of ROS and/or their interaction with membrane pores. Also monoamine oxidase (MAO) inhibitors, like propargylamines, preserve the mitochondria integrity by inhibiting MAO and therefore the production of H2O2 and aldehydes and, as cations, by regulating membrane pores, like BA. As general conclusion, apoptosis is protected by high concentration of BA and/or other cations while it is favoured by ROS produced by AOs or other mechanisms.

Amine oxidases and tumors.

The physiological function in living organisms of amine oxidases, is not completely established but is certainly related to the biogenic amines metabolism and therefore involved in essential processes such as the cell growth and differentiation. A correlation between degree of tumor malignancy and level of AO activity has been reported. The catalytic products of oxidative deamination of amines (hydrogen peroxide and aminoaldehyde) exert a cytotoxic effect and are considered cell growth inhibitors. The biogenic amines in same way could be considered in the cells as both poisons and protectors. A balance of oxidant and antioxidant enzymes appears to be very important in carcinogenesis and cell growth regulation.

Is the catalytic mechanism of bacteria, plant, and mammal copper-TPQ amine oxidases identical?

This short review is mostly concerned with the work carried out in Rome on the copper amine oxidase from bovine serum (BSAO). The first target was the copper oxidation state and its relationship with the organic cofactor. It was found that copper is not reduced on reaction with amines under anaerobic conditions or along the catalytic cycle and that it is not within bonding distance of the quinone cofactor. The copper stability in the oxidised state was supported by BSAO ability to oxidise benzylhydrazine, a slow substrate, in the presence of N,N-diethyldithiocarbamate (DDC) and by the substantial catalytic activity of Co(2+)-substituted BSAO. Parallel work established that only one subunit of the dimeric enzyme readily binds reagents of the carbonyl group. Flexible hydrazides with a long aromatic tail were found to be highly specific inhibitors, suggesting the presence of an extended hydrophobic region at the catalytic site. A study by stopped-flow transient spectroscopy and steady state kinetics led to the formulation of a simplified, yet complete and consistent, catalytic mechanism for BSAO that was compared with that available for lentil seedling amine oxidase (LSAO). As in other copper amine oxidases, BSAO is inactivated by H(2)O(2) produced in the catalytic reaction, while the cofactor is stabilised in its reduced state. A conserved tyrosine hydrogen-bonded to the cofactor might be oxidised.

New perspectives on the role of amine oxidases in physiopathology.

In the paper here presented we summarize some results obtained in our laboratory in the last few years on new structural and functional aspects of some amine oxidases (AOs), which have to be taken into consideration in defining new strategies of controlling the cellular physiopathology. In particular, the ability of Cu-AO purified from vegetal sources or from bovine serum to bind different cellular targets inducing in them conformational as well as chemical modifications are described and the consequences of this interaction on cellular functions are discussed. This is the case of the protective effect of Cu-AO against the damage induced by free radicals, cell enrichment with Cu-AO, induction of cataract and the leukocyte-endothelia interaction. The role of Cu and FAD-amine oxidases related as to the protection or damage of cells is also discussed. In this context the involvement of MAOs in the modulation of the mitochondrial functions and in the induction of apoptosis is described and some aspects of the molecular mechanism of AO inhibition by H(2)O(2) and metronidazole analyzed.

Some new functions of amine oxidases.

Two contrasting topics are examined in this account: the protective actions of amine oxidases (AOs) resulting from the elimination and/or modulation of the levels of polyamines and some biogenic amines, such as histamine, in anaphylactic shock and the cell damaging effect of AOs catabolic products. Other functions of the plasma copper-containing amine oxidase are considered; namely the modification of some proteins by oxidation of their free amino groups, the auto-regulation of the catalytic activity of AOs, the protective effect against free radicals, and the regulation of K(+)-channels.

Modulation of bovine serum amine oxidase activity by hydrogen peroxide.

Bovine serum amine oxidase (BSAO), reduced by excess amine under limited turnover conditions, was over 80% inactivated by H(2)O(2) upon oxygen exhaustion. The UV-Vis spectrum and the reduced reactivity with carbonyl reagents showed that the cofactor topaquinone (TPQ) was stabilized in reduced form. The protein large M(r) (170 kDa) prevented the identification of modified residues by amino acid analyses. Minor changes of the Cu(2+) EPR signal and the formation of a radical at g = 2.001, with intensity a few percent of that of the Cu(2+) signal, unaffected by a temperature increase, suggest that Cu(2+)-bound histidines were not oxidized and the radical was not the Cu(+)-semiquinolamine in equilibrium with Cu(2+)-aminoquinol. It may derive from the modification of a conserved residue in proximity of the active site, possibly the tyrosine at hydrogen-bonding distance of TPQ C-4 ionized hydroxyl. The inactivation reaction appears to be a general feature of copper-containing amine oxidases. It may be part of an autoregulatory process in vivo, possibly relevant to cell adhesion and redox signaling.

Characterization of the haemoglobin-mediated inhibition of the enzymatic activity of bovine serum amine oxidase.

Haemoglobin has been previously identified as responsible for the decreased enzymatic activity of copper bovine serum amine oxidase (BSAO) in suspensions of human or bovine hemolyzed erythrocytes [Marcocci, L., Pietrangeli, P., Befani, O., Mavelli, I., & Mondovi', B. (1991b) Life Chem. Report, 9, 171-177]. This is confirmed by present results on bovine methaemoglobin. Bovine globin and horse skeletal muscle mioglobin showed a similar inhibiting ability, but neither bovine serum albumin nor cytochrome c inhibited BSAO activity under the same experimental conditions. The inhibitory effect of bovine haemoglobin was dependent on pH only at high buffer ionic strength. It was highest in physiological conditions (PBS) where haemoglobin acted as a reversible non competitive inhibitor of BSAO activity, with apparent Ki of 0.5 mM at 37 degrees C. The inhibition was unaffected by partial BSAO deglycosylation (40% of glucidic residues removed) but decreased when haemoglobin lysine groups were derivatised using citraconic anhydride. A possible molecular mechanism underlying the inhibitory effect is discussed.

Inhibitory effect of 1,3,5-triphenyl-4,5-dihydro-(1H)-pyrazole derivatives on activity of amine oxidases.

A new series of 1,3,5-triphenyl-4,5-dihydro-(1H)-pyrazole derivatives was synthesized to ascertain the contribution of substituted phenyl rings present on the 4,5-dihydro-(1H)-pyrazole nucleus to the monoamine oxidases inhibition and bovine serum amine oxidase inhibition. All compounds were tested on bovine brain mitochondria preparation containing flavin-monoamine oxidases and on purified bovine serum amine oxidases, taken as a model of trihydroxyphenylalanine quinone-copper-containing amine oxidases. The 1,3,5-triphenyl-4,5-dihydro-(1H)-pyrazole derivatives showed a good inhibitory activity and belonged to the third generation of monoamine oxidase inhibitors and bovine serum amine oxidase inhibitors which have the advantage of acting through a reversible mode. Furthermore, their activity showed a good degree of selectivity towards the bovine serum amine oxidase inhibition dependent on the substituents present on the phenyl ring at position 5 of the 4,5-dihydro-(1H)-pyrazole.

Protection against apoptosis by monoamine oxidase A inhibitors.

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO-A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

Polyol pathway activation and glutathione redox status in non-insulin-dependent diabetic patients.

The current study aimed to evaluate whether nicotinamide adenine dinucleotide phosphate (NADPH) alteration in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) is responsible for the impaired glutathione (GSH) redox status, and to assess if short-term inhibition of the polyol pathway normalizes NADPH levels and GSH redox status via an amelioration of the NADPH/total NADP (tNADP) ratio. For this purpose, erythrocyte NADPH and GSH levels were measured in 18 NIDDM patients at baseline and then after 1 week of random double-blind assignment to treatment with either tolrestat (an aldose reductase inhibitor, 200 mg daily) (n = 12) or placebo (n = 6). A group of 16 healthy volunteers served as the control. In the basal condition, mean GSH (P < .0001) and NADPH (P < .0001) levels and NADPH/tNADP (P < .0001) and GSH/ glutathione disulfide (GSSG) (P < .005) ratios were lower in NIDDM patients than in control subjects. Tolrestat treatment increased GSH levels (P < .05 v placebo and baseline) and the NADPH/tNADP ratio (P < .05 v placebo and baseline). Interestingly, tolrestat-induced changes in GSH and NADPH levels and in GSH/GSSG and NADPH/tNADP ratios were significant only in patients who showed a decreased NADPH/tNADP ratio at baseline (n = 8). In these latter patients, we also found a direct correlation between percentage increments in GSH levels and NADPH/tNADP ratios after tolrestat treatment (r = .71, P < .05). In conclusion, our findings support the hypothesis that polyol pathway activation decreases NADPH and GSH levels. Accordingly, short-term inhibition of this enzymatic route increased both the GSH level and the NADPH/tNADP ratio. These changes were observable only in the subgroup of patients with an abnormal NADPH/tNADP ratio at baseline. Polyol pathway inhibition could be useful for decreasing oxidative stress in NIDDM.

Extended substrate specificity of serum amine oxidase: possible involvement in protein posttranslational modification.

The capacity of bovine serum amineoxidase (SAO) to oxidize free amino groups of nonconventional substrates, such as polylysine (up to 50 kDa) and some proteins as lysozyme and ribonuclease A, is described. The oxidation was quantified from the amount of H2O2 and NH3 enzymatically produced by SAO. Kinetic analysis indicated a stereospecific preference for L-configuration. Maximal oxidation rate was obtained with poly-L-lysine (9.6 kDa). After 10 h of incubation at 37 degrees C, the poly-L-lysine was partially oxidized generating 1.5 moles of H2O2 by one mole of polylysine. Denatured SAO presented very low oxidation rates with the mentioned substrates.

Enhancement of daunomycin toxicity by the differentiation inducer hexamethylene bisacetamide in erythroleukemia cells.

Cytotoxic effects of daunomycin were investigated upon differentiation of Friend erythroleukemia cells induced with hexamethylene bisacetamide, a process during which a 20-fold increase in the hemoglobin content occurred. Daunomycin proved to be more toxic to differentiated Friend cells than to their undifferentiated counterparts. No changes in the daunomycin uptake rates of the two cell types were detectable. Externally added catalase and desferrioxamine mesylate protected against the additional cytotoxicity of daunomycin in differentiated cells, pointing to hydrogen peroxide and iron ions as mediators of the toxic effect. Daunomycin-dependent, cyanide-insensitive oxygen consumption of control and induced cells did not differ significantly, and the rate of formation of the daunomycin semiquinone radical electron paramagnetic resonance signal was similar in both cell types, indicating that the difference in toxicity was not due to increased drug activation by plasma membrane enzymes. Differentiated cells had a lowered catalase content; the cellular iron content was shown to increase by 2.8-fold upon cell differentiation, with hemoglobin-bound iron being about 50% of the total. Altogether, the results suggest increased intracellular hydrogen peroxide generation mediated by hemoglobin, combined with a decrease in catalase activity and an increase in accessible iron, as responsible for the higher sensitivity to daunomycin shown by differentiated Friend cells. This represents the first experimental system where the increase in anthracycline cytotoxicity upon cell differentiation can be attributed to redox activation and the formation of reactive oxygen species.

Effects of incubation with liposomes at different temperatures on cultured melanoma cells (M14).

A melanoma cell line (M14) was used in order to investigate the effect of hyperthermia on the mechanisms of interaction between liposomes and cultured cells. The treatment was performed by adding different concentrations of multilamellar liposomes (L-alpha-dipalmitoylphosphatidylcholine, stearylamine and cholesterol in the ratio 7:2:1) to cell cultures which were then incubated at 37.0 or 41.5 degrees C for 2 h. The damage induced by liposome treatment in normothermia or hyperthermia was evaluated by determining cell survival and by electron microscopy. When different concentrations of liposomes were used, a dose-dependent impairment of cell survival was observed. An enhancement of the cytotoxic effect was observed when the treatment was performed at 41.5 degrees C. This effect went on even after 24 h from the end of the treatment, but the difference between cells treated in normothermia and hyperthermia was remarkably reduced. The mechanism of the liposome-plasma membrane interaction has been investigated by electron microscopy. Our observations demonstrated that the outer bilayer of the multilamellar liposomes was capable of fusing with the plasma membrane, inducing changes in its fluidity and molecular organization. Following this process the inner liposomal bilayers entered the cell. These effects seemed to be favoured when the treatment was performed under mild hyperthermic conditions, accounting for the synergic cytotoxic action displayed by the liposome-hyperthermia association.

Plasma membrane as a site of redox activation of daunomycin in intact human erythrocytes. Quantitative evaluation of the hydrogen peroxide produced by the membrane with respect to the cytosol.

The relative importance in human red blood cells of the plasma membrane as a site of redox activation of anthracyclines as compared to hemoglobin was evaluated by assaying the H2O2 produced upon exposure to daunomycin. The method of H2O2-dependent irreversible inhibition of catalase (EC 1.11.1.6) activity by 3-amino-1,2,4-triazole was applied to intact erythrocytes, as well as to isolated membranes with added purified catalase. The results obtained indicate a secondary role in daunomycin activation for the plasma membrane from a quantitative point of view, although membrane pathways can be more harmful than cytosolic pathways, especially towards extracellular targets, when the high efficiency of the cytosolic antioxidative defences and the external location of the membrane activation site are considered.

The use of grey literature in health sciences: a preliminary survey.

The paper describes some initiatives in the field of grey literature (GL) and the activities, from 1985, of the Italian Library Association Study Group. The major categories of GL are defined; a survey that evaluates the use of GL by end users in the health sciences is described. References in selected periodicals and databases have been analyzed for the years 1987-1988 to determine the number of articles citing GL, the number of GL citations found in selected periodicals, the various types of GL found, and the number of technical reports cited and their country of origin and intergovernmental issuing organization. Selected databases were also searched to determine the presence of GL during those same years. The paper presents the first results obtained.