The products of six unidentified reading frames of human mitochondrial DNA are precipitated from a mitochondrial lysate by antibodies against highly purified native beef heart NADH-ubiquinone oxidoreductase (complex I). These products are enriched greatly in a human submitochondrial fraction enriched in NADH-Q1 and NADH-K3Fe(CN)6 oxidoreductase activities. We conclude that the six reading frames encode components of the respiratory-chain NADH dehydrogenase.

No abstract

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 inhibitors N 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.Dark-light transitions dramatically affect organ architecture and growth rate during the first stages of plant development. In particular, for a young seedling buried under the soil surface, rapid extension growth of hypogean organs occurs in the dark to reach sunlight. The mesocotyl is devoted to accomplish this important function in maize (Zea mays) and other Gramineae, the growth of this organ being strongly stimulated in the dark, whereas it is inhibited by light as soon as the coleoptile sprouts from the soil surface. This complex photomorphogenic event, mediated by different classes of photoreceptors (Vanderhoef and Briggs, 1978), is thought to be linked to the reduction of indole-3-acetic acid (IAA) supply from the coleoptile to the mesocotyl (Iino, 1982), particularly in its epidermis (Barker-Bridgers et al., 1998). This process causes a tension increase in the tissue, which constrains the growth of the whole organ, the greatest tensile force loading on the outer walls of epidermal cells (Masuda and Yamamoto, 1972; Kutschera and Briggs, 1987; Bret-Harte et al., 1991; Kutschera, 1992).Cell wall yielding properties depend on a finely regulated balance between wall-loosening and -stiffening events. Wall loosening is thought to be mediated by either enzymatic (Cosgrove, 2000; Darley et al., 2001) or chemical agents (Miller, 1986; Fry, 1998; Schopfer, 2001). To this re...

Aim  We discuss biogeographical hypotheses for the Mediterranean lizard species Podarcis and Teira within a phylogenetic framework based on partial mitochondrial DNA sequences. Methods  We derived the most likely phylogenetic hypothesis from our data set (597 aligned positions from the 12S rDNA and phenyl tRNA) under parsimony, distance and maximum likelihood assumptions. Results  The species usually included in Teira do not form a strongly monophyletic clade. In contrast, the monophyly of the genus Podarcis is rather well supported. Seven lineages are identified in the genus; in order of appearance within the tree, these are: the Balearic pityusensis and lilfordi pair, the sicula complex, a Tyrrhenian tiliguerta and raffonei pair, muralis, the Siculo‐Maltese filfolensis and wagleriana pair, the Balkan group (erhardi, peloponnesiaca, milensis, melisellensis and taurica), and the Ibero‐Maghrebian group (bocagei, atrata, hispanica and vaucheri). Conclusions  The origin of the three European genera of lacertid assayed (Lacerta, Teira and Podarcis) is hypothesized to have occurred in the Oligocene. For Podarcis, a possible scenario of a Miocene diversification is derived from the sequence data, and the zoogeography of the lineages are discussed in relation to the palaeogeography of the Mediterranean. It is hypothesized that in the early history of the genus the main lineages separated by rapid, numerous and close events that produced a starting point very similar to a polytomy, hard to resolve by parsimony analysis of the data set.

‘Cryptic’ species are an emerging biological problem that is broadly discussed in the present study. Recently, a cryptic species definition was suggested for those species which manifest low morphological, but considerable genetic, disparity. As a case study we present unique material from a charismatic group of nudibranch molluscs of the genus Trinchesia from European waters to reveal three new species and demonstrate that they show a dual nature: on one hand, they can be considered a ‘cryptic’ species complex due to their overall similarity, but on the other hand, stable morphological differences as well as molecular differences are demonstrated for every species in that complex. Thus, this species complex can equally be named ‘cryptic’, ‘pseudocryptic’ or ‘non-cryptic’. We also present evidence for an extremely rapid speciation rate in this species complex and link the species problem with epigenetics. Available metazoan-wide data, which are broadly discussed in the present study, show the unsuitability of a ‘cryptic’ species concept because the degree of crypticity represents a continuum when a finer multilevel morphological and molecular scale is applied to uncover more narrowly defined species making the ‘cryptic’ addition to ‘species’ redundant. Morphological and molecular methods should be applied in concordance to form a fine-scale multilevel taxonomic framework, and not necessarily implying only an a posteriori transformation of exclusively molecular-based ‘cryptic’ species into morphologically-defined ‘pseudocryptic’ ones. Implications of the present study have importance for many fields, including conservation biology and fine-scale biodiversity assessments.

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 dinucleotidecontaining 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 calculated M 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 into Escherichia 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 ϳ68,000) has K m and k cat values of 90 M 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.Polyamine oxidase (PAO), 1 a flavin adenine dinucleotide (FAD)-containing enzyme, catalyzes the oxidation of polyamines at the secondary amino group, giving different products according to the organism considered. In particular, vertebrate PAOs (EC 1.5.3.11) participate in the interconversion metabolism of polyamines, converting N 1 -acetyl derivatives of spermine (N 1 -acetylSpm) and spermidine (N 1 -acetylSpd) into Spd and putrescine, respectively, plus 3-aminopropanal and H 2 O 2 , (1-3). PAOs with similar characteristics occur in methylotrophic yeasts (4, 5) and amoebae (6). On the contrary, plant (1), bacterial (7), and protozoan (8) PAOs oxidize spermidine and spermine to 4-aminobutanol or N-(3-aminopropyl)-4-aminobutanol, respectively, plus 1,3-diaminopropane and H 2 O 2 . As these compounds cannot be converted directly to other polyamines, this class of PAOs generally is considered to be involved in the terminal catabolism of polyamines. Since PAOs play a crucial role in polyamine catabolism, these enzymes are important drug targets, and in fact, it has been shown that a number of polyamine analogues have an antitumor effect in different cell lines (9 -12).As compared with large and detailed investigations on plant PAOs (1, 13-22), only little attention has been devoted to the animal counterpart (2,(23)(24)(25)(26)(27)(28). Recently, Wang et al. (29) and Vujcic et al. (30) have reported the cloning and characterization of novel mammalian PAO enzymes capable of oxidizing preferentially Spm and for this reason named spermine oxidase (SMO) (30). In ...

The polyamines putrescine (put), spermidine (spd), and spermine (spm) are natural aliphatic polycations ubiquitous in living organisms and essential for cell growth, proliferation, and differentiation. For a long time the polyamine biosynthetic pathway has been considered to be the unique target for antineoplastic therapy [1][2][3]. The importance of the polyamine catabolic pathway has recently been re-evaluated, as its involvement in determining the cell response to antitumour polyamine analogues has been demonstrated [4].In animals, polyamine catabolism is a recycling pathway that converts spm to spd and spd to put, with the production of toxic aldehydes and H 2 O 2 . Polyamine catabolism is achieved via the concerted action 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,N 1 -bis(2,3-butadienyl)-1,4-butanediamine (MDL72527) is reported. Interestingly, 1,12-Diaminododecane and G3 behave as specific inhibitors of mPAO, values of K i 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.Abbreviations

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, K m and V max 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.Keywords: barley; enzyme isoform; maize; polyamine oxidase; subcellular localization.Polyamine oxidase (PAO), a FAD-containing enzyme, catalyses the oxidation of polyamines at the secondary amino group, yielding different products in different taxa. In particular, vertebrate PAOs (EC 1.5.3.11), which are localized in peroxisomes, efficiently transform N 1 -acetyl derivatives of spermidine and spermine into putrescine and spermidine, respectively, plus acetamidopropanal and H 2 O 2 , thus participating in the interconversion metabolism of polyamines [1±3]. PAOs with similar characteristics occur in methylotrophic yeasts [4,5] and amoebae [6]. On the other hand, plant [1], bacterial [7] and protozoal [8] PAOs oxidize spermidine and spermine to 4-aminobutanal and N-(3-aminopropyl)-4-aminobutanal, respectively, plus 1,3-diaminopropane and H 2 O 2 . As these compounds cannot be converted directly to other polyamines, this class of PAO is considered to be involved in the terminal catabolism of polyamines. Plant PAOs, which apparently occur mainly in the cell wall of monocots, have been purified and partially characterized from a few species [1,9,10]. PAO from maize (MPAO), the most studied member of this enzyme class, is a 53-kDa monomeric glycoprotein containing one molecule of FAD [2,7]. Recently, its complete amino-acid sequence has been determined by a combined approach of protein and cDNA sequencing [11], and its crystal structure has been solved to a resolution of 1.9 A Ê [12]. MPAO has been found mainly in the apoplast by means of biochemical and immu...