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Biomedical subjects

R Federico

Publications and source records attributed to R Federico.

At least 19 recordsLinked to original sources
PubMed2003

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.

Journal Article↗
PubMed2001

Structural bases for inhibitor binding and catalysis in polyamine oxidase.

Polyamine oxidase (PAO) carries out the FAD-dependent oxidation of the secondary amino groups of spermidine and spermine, a key reaction in the polyamine catabolism. The active site of PAO consists of a 30 A long U-shaped catalytic tunnel, whose innermost part is located in front of the flavin ring. To provide insight into the PAO substrate specificity and amine oxidation mechanism, we have investigated the crystal structure of maize PAO in the reduced state and in complex with three different inhibitors, guazatine, 1,8-diaminooctane, and N(1)-ethyl-N(11)-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm). In the reduced state, the conformation of the isoalloxazine ring and the surrounding residues is identical to that of the oxidized enzyme. Only Lys300 moves away from the flavin to compensate for the change in cofactor protonation occurring upon reduction. The structure of the PAO.inhibitor complexes reveals an exact match between the inhibitors and the PAO catalytic tunnel. Inhibitor binding does not involve any protein conformational change. Such lock-and-key binding occurs also in the complex with CHENSpm, which forms a covalent adduct with the flavin N5 atom. Comparison of the enzyme complexes hints at an "out-of-register" mechanism of inhibition, in which the inhibitor secondary amino groups are not properly aligned with respect to the flavin to allow oxidation. Except for the Glu62-Glu170 pair, no negatively charged residues are involved in the recognition of substrate and inhibitor amino groups, which is in contrast to other polyamine binding proteins. This feature may be exploited in the design of drugs specifically targeting PAO.

Binding, Competitive↗
PubMed2001

Analysis of the distribution of copper amine oxidase in cell walls of legume seedlings.

Copper-containing amine oxidase (CuAO) has been proposed to play a role in H2O2 production in plant cell walls during cell development and in response to pathogen attack. We have compared the localisation of CuAO in pea (Pisum sativum L.), lentil (Lens culinaris M.) and chick pea (Cicer arietinum L.) grown under different light conditions, using both immuno- and histochemical techniques. The enzyme was detected by indirect immunofluorescence in the cell walls of parenchyma tissues of etiolated pea and lentil plants and was particularly abundant at intercellular spaces. Upon de-etiolation, CuAO largely disappeared from cortical cell walls except in the region of intercellular spaces. In the apical internode of light-grown seedlings, CuAO occurred mainly in cortical cell walls and, to some extent, in cell walls of xylem vessels. In both the elongation zone and mature regions of roots, CuAO was restricted to cortical cell walls and some cell junctions close to the meristem. Extensin epitopes co-localised to intercellular spaces of the cortex in de-etiolated pea, indicating that CuAO may have a role in cell wall strengthening at intercellular spaces. In chick pea, the localisation of the enzyme varied between different cultivars that have differing susceptibility to the fungus Ascochyta rabiei. In a susceptible cultivar Calia, immunogold labelling localised CuAO to cell walls of the cortex, as in lentil and pea, while in a resistant cultivar Sultano, it was most abundant in xylem vessels and, in light-grown plants, in the epidermis. These expression patterns are discussed with regard to the possible functions of amine oxidase in cell growth, cell differentiation and pathogen resistance.

Amine Oxidase (Copper-Containing)↗
PubMed2001

Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen.

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

Allergens↗
PubMed2001

A monoclonal antibody specific for a carbohydrate epitope recognizes an IgE-binding determinant shared by taxonomically unrelated allergenic pollens.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Allergens↗
PubMed2001

A barley polyamine oxidase isoform with distinct structural features and subcellular localization.

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.

Amino Acid Sequence↗
PubMed2001

Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study.

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.

Agmatine↗
PubMed2000

Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug.

Clonidine, clinically used in the treatment of hypertension, is a central alpha(2)-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity. However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K(i) for clonidine binding to NOS-I increased (from [7. 9 +/- 0.4] x 10(-5) M to [8.0 +/- 0.4] x 10(-3) M) as BH(4) concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees. In addition, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Animals↗
PubMed1999

A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase.

BACKGROUND: Polyamines are essential for cell growth and differentiation; compounds interfering with their metabolism are potential anticancer agents. Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary amino groups of spermine and spermidine. RESULTS: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Angstroms. PAO from Zea mays contains two domains, which define a remarkable 30 Angstrom long U-shaped catalytic tunnel at their interface. The structure of PAO in complex with the inhibitor MDL72527 reveals the residues forming the catalytic machinery and unusual enzyme-inhibitor CH.O H bonds. A ring of glutamate and aspartate residues surrounding one of the two tunnel openings contributes to the steering of the substrate towards the inside of the tunnel. CONCLUSIONS: PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.

Amino Acid Sequence↗
PubMed1998

Crystallization and preliminary X-ray analysis of polyamine oxidase from Zea mays L.

Polyamine oxidase catalyses the oxidation of the secondary amino group of spermine, spermidine and their acetyl derivatives. The enzyme plays an important role in the regulation of polyamine intracellular concentration and is a member of the family of flavin-containing amine oxidases. Crystals of maize polyamine oxidase have been grown by the hanging-drop vapour-diffusion technique. The crystals are in hexagonal space group P6122 (or P6522) with cell dimensions a = b = 184.6, c = 280.9 A. A native data set has been collected to 2.7 A resolution at a synchrotron radiation source.

Binding Sites↗
PubMed1998

Developmentally and wound-regulated expression of the gene encoding a cell wall copper amine oxidase in chickpea seedlings.

A chickpea cDNA encoding a cell wall copper amine oxidase (CuAO) was cloned and characterised. The 2010 bp open reading frame encodes a protein of 76.5 kDa which shares significant primary structure homology with other known CuAOs. Southern blot analysis indicates that in chickpea CuAO is encoded by a single gene or a small gene family. This cDNA was essential for studying the role of CuAO during seedling development and wound healing in chickpea seedlings. CuAO transcript level and activity were modulated during seedling development in parallel with cell maturation. Moreover, mechanical wounding induced a rapid increase of CuAO mRNA accumulation and enzyme activity which remained high during the wound-healing process. Aminoguanidine, a specific CuAO inhibitor, decreased the deposition of lignin-suberin barrier along the lesion. CuAO may be a limiting factor in H2O2 production in the cell wall of chickpea seedlings and its expression seems to integrate with the remodelling of plant cell wall occurring during ontogenesis and wound healing.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Maize polyamine oxidase: primary structure from protein and cDNA sequencing.

The first complete amino acid sequence of a flavin-containing polyamine oxidase was solved by a combined approach of nucleotide and peptide sequence analysis. A cDNA of 1737 bp, isolated from maize seedlings by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends strategies, was cloned and its sequence determined. This cDNA contains information for a polypeptide chain of 500 amino acids. Its amino-terminal sequence shows the typical features of secretion signal peptides. The primary structure of the mature protein was independently confirmed by extensive amino acid sequencing. Structural relationships with flavin-containing monoamine oxidases are also discussed.

Amino Acid Sequence↗
PubMed1998

Competitive inhibition of Lens culinaris L. copper amine oxidase by amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenylindole and gabexate mesylate: a comparative study.

The competitive inhibition of Lens culinaris L. copper amine oxidase by amiloride (Ki = 4.1 x 10(-4) M), p-aminobenzamidine (Ki = 6.0 x 10(-4) M), clonidine (Ki = 5.0 x 10(-4) M), 4',6-diamidino-2-phenylindole (DAPI; Ki = 1.9 x 10(-5) M) and gabexate mesylate (Ki = 2.5 x 10(-4) M) has been investigated, at pH 7.0 and 25 degrees C. The affinity of p-aminobenzamidine, clonidine and DAPI for plant and mammalian copper amine oxidase is closely similar. However, values of Ki for amiloride and gabexate mesylate binding to swine kidney copper amine oxidase are lower than those observed for inhibitor binding to Lens culinaris L. cooper amine oxidase. Thus, amiloride and gabexate mesylate may represent useful model compounds for the development of selective inhibitors of mammalian copper amine oxidase, which may be important in view of the potential use of plant copper amine oxidase as drugs.

Amiloride↗
PubMed1997

Competitive inhibition of swine kidney copper amine oxidase by drugs: amiloride, clonidine, and gabexate mesylate.

Competitive inhibition of swine kidney copper amine oxidase by diuretic, antihypertensive, and anticoagulant drugs, amiloride, clonidine, and gabexate mesylate, respectively, is reported. The affinity of these compounds for swine kidney copper amine oxidase is similar to that observed for inhibitor binding to nitric oxide synthase and trypsin-like serine proteinases. This finding suggests that amiloride, clonidine, and gabexate mesylate should be administrated under careful control, since enzyme cross-inhibition may occur also in vivo.

Agmatine↗