The Osteological Collection of the University of Cagliari: From Early Neolithic to Modern Age.

The Osteological Collection described in this paper is located at the Anthropology Laboratory annexed to the Sardinian Museum of Anthropology and Ethnography of the University of Cagliari. It has been created in 1953 by Carlo Maxia and comprises a large number of skeletal remains. At present the Collection consists of 11,854 human bones and continues to grow. The remains belong to different periods, beginning from the Early Neolithic and continuing to the Modern Age. The aim of this work is to provide information on the composition of this collection after the reorganization carried out in the last years.

Inhibitory interneuron classes express complementary AMPA-receptor patterns in macaque primary visual cortex.

Glutamate receptors mediate excitatory neurotransmission. A very prevalent type of glutamate receptor in the neocortex is the AMPA receptor (AMPAR). AMPARs mediate fast synaptic transmission and their functionality depends on the subunit composition. In primary visual cortex (area V1), the density and subunit composition of AMPARs differ among cortical layers and among cell types. The AMPARs expressed by the different types of inhibitory interneurons, which are crucial for network function, have not yet been characterized systematically. We investigated the distribution of AMPAR subunits in macaque V1 for three distinct subpopulations of inhibitory interneurons: parvalbumin-immunoreactive (PV-IR) interneurons, calbindin-immunoreactive (CB-IR) interneurons, and calretinin-immunoreactive (CR-IR) interneurons. We found that PV-IR cells, which have previously been identified as fast spiking, show high expression of the GluA2 and GluA3 subunits. In contrast, CB-IR and CR-IR cells, which tend to be intermediate spiking, show high expression of the GluA1 and GluA4 subunits. Thus, our data demonstrate that the expression of AMPARs divides inhibitory interneurons in macaque V1 into two categories that are compatible with existing classification methods based on calcium-binding proteins and firing behavior. Moreover, our findings suggest new approaches to target the different inhibitory interneuron classes pharmacologically in vivo.

Craniofacial morphometric variation and the biological history of the peopling of Sardinia.

The aim of this work is to explore the pattern of craniofacial morphometric variation and the relationships among five prehistoric Sardinian groups dated from Late Neolithic to the Nuragic Period (Middle and Late Bronze Age), in order to formulate hypotheses on the peopling history of Sardinia. Biological relationships with coeval populations of central peninsular Italy were also analysed to detect influences from and towards extra-Sardinian sources. Furthermore, comparison with samples of contemporary populations from Sardinia and from continental Italy provided an indication of the trend leading to the final part of the peopling history. Finally, Upper Palaeolithic and Mesolithic samples were included in the analyses to compare the prehistoric Sardinians with some of their potential continental ancestors. The analysis is based on multivariate techniques including Mahalanobis D(2) distance, non-parametric multidimensional scaling (MDS) and principal component analysis (PCA). The results showed the tendency to progressive differentiation between Sardinian groups and peninsular Italian groups, with the possible exception of a discontinuity showed by the Bonnànaro (Early Bronze Age) Sardinian sample. Several aspects of the morphological results were found to agree with the current genetic evidence available for the present-day Sardinian population and a Nuragic sample: (1) biological divergence between the Sardinian and peninsular Italian populations; (2) similarity/continuity among Neolithic, Bronze Age and recent Sardinians; (3) biological separation between the Nuragic and Etruscan populations; (4) contribution of a Palaeo-Mesolithic gene pool to the genetic structure of current Sardinians.

Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat.

The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.

Catalase and antiquitin from Euphorbia characias: two proteins involved in plant defense?

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant's protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

Purification and properties of a nucleotide pyrophosphatase from lentil seedlings.

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 +/- 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.

Arginine and ornithine oxidation catalyzed by lentil seedling copper-amine oxidase.

The oxidation of L-ornithine and L-arginine catalyzed by lentil (Lens esculenta) seedling copper-amine oxidase has been investigated by polarographic techniques, optical spectroscopy, and capillary electrophoresis. Both L-ornithine and L-arginine were found to be poor substrates for lentil amine oxidase. L-Ornithine was oxidized to glutamate-5-semialdehyde and ammonia, in similar manner as usual substrates. Glutamate-5-semialdehyde spontaneously cyclizes to delta1-pyrroline-5-carboxylic acid. Arginine is oxidized by an unusual mechanism yielding glutamate-5-semialdehyde, ammonia, and urea as reaction products.

Interaction of pig kidney and lentil seedling copper-containing amine oxidases with guanidinium compounds.

The effect of guanidinium compounds on the catalytic mechanism of pig kidney and lentil seedling amine oxidases has been investigated by polarographic techniques and spectroscopy. Guanidine does not inhibit the lentil enzyme and is a weak inhibitor for pig kidney amine oxidase (Ki=1 mM), whereas aminoguanidine is an irreversible inhibitor of both enzymes, with a Ki value of 10(-6) M. 1,4-Diguanidino butane (arcaine) is a competitive inhibitor for both pig and lentil amine oxidases. Amiloride is a competitive inhibitor for pig enzyme, but upon prolonged incubation with this drug the enzyme gradually loses its activity in an irreversible manner.

Effect of metal substitution in copper amine oxidase from lentil seedlings.

The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k(c) value is different from that of native or Cu-fully-reconstituted enzyme, while K(m) is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

CuI-semiquinone radical species in plant copper-amine oxidases.

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

Intermediates in the catalytic cycle of lentil (Lens esculenta) seedling copper-containing amine oxidase.

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. gamma-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.

Inhibition of copper amine oxidase by haloamines: a killer product mechanism.

The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme.

Cysteamine oxidation by lentil seedling amine oxidase.

Cysteamine is oxidatively deaminated by lentil amine oxidase. It shows saturation kinetic K(m) = 9 x 10(-4) M like other substrates, but the aldehyde produced leads to loss of enzyme activity, which is restored by dialysis. When putrescine is the substrate of the amine oxidase cysteamine behaves like a competitive inhibitor, and shows Ki = 5 x 10(-5) M. The possible involvement of the oxidation of cysteamine and the inhibitory effects of thioacetaldehyde in the cystamine oxidation by amine oxidase is discussed.

Characterization of a cyclic compound formed after spermine oxidation by lentil amine oxidase.

Spermine is a substrate of lentil seedling amine oxidase and is oxidized at terminal amino groups to a dialdehyde: 2 mol of hydrogen peroxide and two mol of ammonia per mol of spermine are formed. In the presence of high amounts of spermine, the aldehydic groups formed upon oxidation of spermine by the enzyme, may react with primary amino groups of free spermine leading to the formation of aromatic pyrimidinic ring after beta-elimination at secondary amino groups.

Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase.

Copper amine oxidase from lentil seedlings was shown to be able to catalyze the oxidative deamination of the indoleamines tryptamine, 5-hydroxytryptamine, and 5-methoxytryptamine. These compounds showed saturation kinetics with Km values as normal substrates, but their oxidation led to irreversible loss of enzyme activity suggesting a covalent interaction with the enzyme, most probably through its cofactor 6-hydroxydopa (2,4,5-trihydroxyphenylalanine). These indoleamines acted as irreversible inhibitors of the enzyme only in the absence of oxygen but they brought about changes in the electronic spectra of the enzyme both in aerobiosis and in anaerobiosis. This study reports on the mechanism by which these compounds inhibit lentil amine oxidase which involves first the oxidation of indoleamines bound to 6-hydroxydopa followed by the formation of an irreversible covalent derivative. The same inhibitory mechanism could possibly lead to inactivation of mammalian amine oxidases involved in serotonin neurotransmitter metabolism in conditions of ischemia or hypoxia.

Substrate specificity of lentil seedling amine oxidase.

Copper diamine oxidase from lentil (Lens culinaris) seedlings was shown to be able to catalyze the oxidative deamination of a wide range of aliphatic and aromatic monoamine compounds, including some amino acids. Although the catalytic efficiencies were only 1-3% of that measured with the diamine substrate putrescine, they were still comparable to those of specialized monoamine oxidases. In particular, the lentil enzyme oxidized benzylamine and histamine with K(m) and Vmax values similar to those found for the mammalian enzymes benzylamine oxidase and histaminase. Cysteamine was found to be a substrate of the enzyme, whereas hypotaurine and taurine were found to be neither substrates nor inhibitors of the enzyme. Quite unexpectedly the amino acids L-ornithine and L-lysine were oxidized by lentil enzyme, and beta-alanine and gamma-aminobutyric acid were oxidized only at high concentrations of enzyme. These results suggest that enzymes normally classified as diamine oxidases could in fact have a more diversified role in metabolism than recognized so far.

The reaction mechanism of copper amine oxidase: detection of intermediates by the use of substrates and inhibitors.

Intermediate states in the catalytic mechanism of lentil copper amine oxidase have been investigated by ESR and optical spectroscopy. Using highly purified apo- and holoenzyme in combination with a poor substrate and a range of inhibitors, under both aerobic and anaerobic conditions, the single steps of the reaction mechanism can be slowed down or 'frozen' completely. In this way, a sequence of six intermediate species in the catalytic cycle has been established. Oxidative deamination of p-(dimethylamino)benzylamine is 5 x 10(5) times slower than for putrescine; the rate-limiting step is shown to be release of the aldehyde product. This process is not affected in the apoenzyme, but subsequent intramolecular electron transfer to form the characteristic free radical intermediate is completely blocked, and the apoenzyme is trapped as an aminoresorcinol species. Classic hydrazine and hydrazide inhibitors bind to the 6-hydroxydopa cofactor in the same way as active substrates, but rearrangements lead to formation of stable intermediate adducts at the step preceding release of aldehyde. The semicarbazide-6-hydroxydopa adduct is shown to bind simultaneously to Cu(II), providing the first direct evidence for localization of 6-hydroxydopa close to the copper site.

On the use of 2,4,5-trihydroxyphenethylamine as a rapid method for laccase quantification.

The sensitivity of the autoxidation of 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine) to increase by laccase was studied. The autoxidation of 6-hydroxydopamine proceeds by a free radical chain reaction involving O2- and produces the corresponding chromogen 6-hydroxydopaminequinone and hydrogen peroxide. The ability of laccase to increase the autoxidation of 6-hydroxydopamine at pH 5.5 has been used as sensitive assay for this enzyme. The results are tabulated and discussed.

Evidence for alpha-proton abstraction and carbanion formation involving a functional histidine residue in lentil seedling amine oxidase.

Lentil seedling amine oxidase catalyzes the oxidation of putrescine and in the presence of tetranitromethane gives rise to the formation of nitroform anion. The initial rate of substrate and enzyme-dependent nitroform production is linearly related to the functional active site content and is proportional to the tetranitromethane concentration. Diethylpyrocarbonate modifies two histidyl residues on the lentil amine oxidase. Incubation of the enzyme with diethylpyrocarbonate at 25 degrees C and pH 7.0 irreversibly inhibits enzyme activity by a pseudo first-order kinetics process. The data obtained are consistent with the enzyme-dependent abstraction of an alpha-proton from the substrate to form an intermediate enzyme bound carbanion and indicate a functional role for histidine in lentil amine oxidase catalysis consistent with that of a general base in proton abstraction.

Lentil seedling amine oxidase: interaction with carbonyl reagents.

Carbonyl reagents containing a primary amino group were covalently bound by lentil seedling amine oxidase to resolve conflicting data for both the number of functional active sites in the dimeric enzyme. Active site titration of highly purified enzyme samples with all the carbonyl reagents extrapolates to 1 mol of inhibitor/mol of enzyme subunit indicating the presence of a cofactor at each enzyme subunit. This result is at variance with numerous previous reports of only one functional cofactor for enzyme dimer in copper amine oxidase.