Paolo Mariottini

Polyamine oxidase, a hydrogen peroxide-producing enzyme, is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl.

Exogenously supplied auxin (1-naphthaleneacetic acid) inhibited light-induced activity increase of polyamine oxidase (PAO), a hydrogen peroxide-producing enzyme, in the outer tissues of maize (Zea mays) mesocotyl. The same phenomenon operates at PAO protein and mRNA accumulation levels. The wall-bound to extractable PAO activity ratio was unaffected by auxin treatment, either in the dark or after light exposure. Ethylene treatment did not affect PAO activity, thus excluding an effect of auxin via increased ethylene biosynthesis. The auxin polar transport inhibitorsN 1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid caused a further increase of PAO expression in outer tissues after light treatment. The small increase of PAO expression, normally occurring in the mesocotyl epidermis during plant development in the dark, was also inhibited by auxin, although to a lesser extent with respect to light-exposed tissue, and was stimulated by N 1-naphthylphthalamic acid or 2,3,5-triiodobenzoic acid, thus suggesting a complex regulation of PAO expression. Immunogold ultrastructural analysis in epidermal cells revealed the association of PAO with the secretory pathway and the cell walls. The presence of the enzyme in the cell walls of this tissue greatly increased in response to light treatment. Consistent with auxin effects on light-induced PAO expression, the hormone treatment inhibited the increase in immunogold staining both intraprotoplasmically and in the cell wall. These results suggest that both light and auxin finely tune PAO expression during the light-induced differentiation of the cell wall in the maize mesocotyl epidermal tissues.

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.

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculatedM r of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms intoEscherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N 1-acetylpolyamines. The purified recombinant-tagged form enzyme (M r∼68,000) has K m and k catvalues of 90 μm and 4.5 s−1, respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.

Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae).

Meloid beetles are well characterised by both morphological and biological features. Previous phylogenetic hypotheses based on morphological characters assumed the repeated parallel evolution of complex biological novelties. In this work relationships among several taxa of the four subfamilies and almost all tribes representing meloid diversity are examined by using mitochondrial (16S) and nuclear (ITS2) DNA sequences, in 25 genera (using Anthicidae as outgroup). Secondary structure of 16S and ITS2 rRNAs were modelled. ITS2 structure represents a synapomorphic condition for the family and informative characters at the tribal level. Phylogenetic hypotheses based on separate and combined analysis of the 16S and ITS2 rDNA sequences, and morpho-biological characters were tested, and compared with previous morphological classifications. Molecular dating allowed an outline of the main steps of the evolutionary history of Meloidae, which evolved during Early Cretaceous and then radiated considerably with the adoption of hypermetaboly and parasitic behaviour, and with repeated, parallel evolution of larval phoresy on its hosts.

Inhibition of polyamine and spermine oxidases by polyamine analogues

Polyamine oxidase (PAO) and spermine oxidase (SMO) are involved in the catabolism of polyamines – basic regulators of cell growth and proliferation. The discovery of selective inhibitors of PAO and SMO represents an important tool in studying the involvement of these enzymes in polyamine homeostasis and a starting point for the development of novel antineoplastic drugs. Here, a comparative study on murine PAO (mPAO) and SMO (mSMO) inhibition by the polyamine analogues 1,8‐diaminooctane, 1,12‐diaminododecane, N‐prenylagmatine (G3), guazatine and N,N1‐bis(2,3‐butadienyl)‐1,4‐butanediamine (MDL72527) is reported. Interestingly, 1,12‐Diaminododecane and G3 behave as specific inhibitors of mPAO, values of Ki for mPAO inhibition being lower than those for mSMO inactivation by several orders of magnitude. The analysis of molecular models of mPAO and mSMO indicates a significant reduction of the hydrophobic pocket located in maize PAO (MPAO) at the wider catalytic tunnel opening. This observation provides a rationale to explain the lower affinity displayed by G3, guazatine and MDL72527 for mPAO and mSMO as compared to MPAO. The different behaviour displayed by 1,12‐diaminododecane towards mPAO and mSMO reveals the occurrence of basic differences in the ligand binding mode of the two enzymes, the first enzyme interacting mainly with substrate secondary amino groups and the second one with substrate primary amino groups. Thus, the data reported here provide the basis for the development of novel and selective inhibitors able to discriminate between mammalian SMO and PAO activities.

Spermine Metabolism and Anticancer Therapy

The natural polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM) are ubiquitous constituents of eukaryotic cells. The increase of PA in malignant and proliferating cells attracted the interest of scientists during last decades, addressing PA depletion as a new strategy to inhibit cell growth. Selective enzyme inhibitors were developed for decreasing PA metabolism and to act as chemotherapeutic anticancer agents. Indeed, the complexity of the PA homoeostasis overcomes the PA perturbation by a single enzyme to take effect therapeutically. Recently, an increasing interest has been posed on spermine-oxidase (SMO), the only catabolic enzyme able to specifically oxidise SPM. Interestingly, the absence of SPM is compatible with life, but its accumulation and degradation is lethal. Augmented SMO activity provokes an oxidative stress rendering cells prone to die, and appears to be important in the cell differentiation pathway. Extra-cellular SPM is cytotoxic, but its analogues are capable of inhibiting cell growth at low concentrations, most likely by intracellular SPM depletion. These pivotal roles seem to evoke the biological processes of stress response, wherein balance is mandatory to live or to die. Thus, altering SPM metabolism could allow a multi-tasking therapeutic strategy, addressed not only to inhibit PA metabolism. Several tetramines are presently in early phases (I and II) of clinical trials, and it will be a matter of a few more years to understand whether SPM-related therapeutic approaches would be of benefit for composite treatment protocols of cancer.

Isolation and characterization of three polyamine oxidase genes from Zea mays.

Isolation and sequencing of three genes, MPAO1, MPAO2 and MPAO3, coding for polyamine oxidase (PAO) from maize (Zea mays) are reported here. Gene organization is extremely conserved among these copies, being composed of eight exons and seven introns. Furthermore, these genes encode for a protein of an almost identical amino acid sequence. These data suggest that the three MPAO copies have been derived from gene duplication of a common ancestor gene. Long inverted repeat sequences, also present in other maize genes, have been found within the second intron. Promoter sequences of MPAO1 and MPAO2 genes have been analysed for putative cis-acting elements. According to genomic Southern blot analysis, the MPAO gene family in maize and other monocots is represented by a small number of copies. Northern and western blot analysis have revealed a tissue-specific accumulation of both MPAO mRNA and protein.

A role for spermine oxidase as a mediator of reactive oxygen species production in HIV-Tat-induced neuronal toxicity

Chronic oxidative stress, which occurs in brain tissues of HIV-infected patients, is involved in the pathogenesis of HIV-associated dementia. Oxidative stress can be induced by HIV-1-secreted proteins, either directly or indirectly through the release of cytotoxic factors. In particular, HIV-1 Tat is able to induce neuronal death by interacting with and activating the polyamine-sensitive subtype of the NMDA receptor (NMDAR). Here, we focused on the role of polyamine catabolism in Tat-induced oxidative stress in human neuroblastoma (SH-SY5Y) cells. First, Tat was found to induce reactive oxygen species production and to affect cell viability in SH-SY5Y cells, these effects being mediated by spermine oxidase (SMO). Second, Tat was observed to increase SMO activity as well as decreasing the intracellular spermine levels. Third, Tat-induced SMO activation was completely prevented by the NMDAR antagonist MK-801, clearly indicating an involvement of NMDAR stimulation. Finally, pretreatment of cells with N-acetylcysteine, a scavenger of H₂O₂, and with MK-801 was able to completely inhibit reactive oxygen species formation and to restore cell viability. Altogether, these data strongly suggest a role for polyamine catabolism-derived H₂O₂ in neurotoxicity as elicited by Tat-stimulated NMDAR.

Molecular phylogenetics of the Italian Podarcis lizards (Reptilia, Lacertidae)

Abstract Phylogenetic relationships within the Italian species of the lacertid genus Podarcis were examined by parsimony analysis of mitochondrial DNA sequences from the genes encoding the small ribosomal RNA and the phenyl transfer RNA. Lacerta viridis was used as outgroup and Teira dugesii was also included in the ingroup. The 80 phylogenetically informative positions produced four most parsimonious trees, with the Italian Podarcis split into three groups: the first comprised P. filfolensis, P. melisellensis. P. wagleriaria, P. muralis, and P. raffonei, the second P. sicula with its various subspecies. The third lineage was that of P. tiliguerta, whose relationships were resolved as more closely related to the first clade, when weighting transversion three times transitions. Bootstrap analyses on a subset of sequences representing all species herein studied, supported the results from the larger dataset. The present results are only partly in agreement with previous hypotheses based on morphology, immun...

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure–function relationships, SMO enzyme–substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme–substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.