Polyamines are aliphatic amines with low molecular weight that are widely recognized as one of the most important cancer biomarkers for early diagnosis and treatment. The goal of the work herein presented is the development of a rapid and simple method for the quantification of free polyamines (i.e., putrescine, cadaverine, spermidine, spermine) and N-monoacetylated polyamines (i.e., N-Acetylspermidine, N-Acetylspermidine, and N-Acetylspermine) in human urine. A preliminary derivatization with propyl chloroformate combined with the use of solid phase microextraction (SPME) allowed for an easy and automatable protocol involving minimal sample handling and no consumption of organic solvents. The affinity of the analytes toward five commercial SPME coatings was evaluated in univariate mode, and the best result in terms of analyte extraction was achieved using the divinylbenzene/carboxen/polydimethylsiloxane fiber. The variables affecting the performance of SPME analysis were optimized by the multivariate approach of experimental design and, in particular, using a central composite design (CCD). The optimal working conditions in terms of response values are the following: extraction temperature 40 °C, extraction time of 15 min and no addition of NaCl. Analyses were carried out by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) in selected reaction monitoring (SRM) acquisition mode. The developed method was validated according to the guidelines issued by the Food and Drug Administration (FDA). The satisfactory performances reached in terms of linearity, sensitivity (LOQs between 0.01 and 0.1 ?g/mL), matrix effect (68-121%), accuracy, and precision (inter-day values between -24% and +16% and in the range 3.3-28.4%, respectively) make the proposed protocol suitable to be adopted for quantification of these important biomarkers in urine samples.

The effects of UV-B irradiation and Biomin (a natural substance extracted from coal with active ingredients of humic acids) on the content of endogenous polyamines spermine, spermidine and putrescine, and free amino acids in shoots and roots of young triticale seedlings were investigated. Biomin was added to the nutrient medium 3 days prior to UVB irradiation. The seedlings were treated with 7.7 kJ m(-2) day(-1) UV-B light for 4 days. The exposure to UV-B increased total free amino acids, while Biomin application alone did not affect considerably their content. The treatment with UV-B or Biomin alone provoked augmentation of conjugated and bound polyamine (PA) fractions. Data suggest that Biomin alleviates the negative consequences of UV-B stress, manifested by the normalized amino acid and polyamine amounts in the UV-B + Biomin-treated plants. This study demonstrates the protective effect of Biomin on triticale plants against UV-B irradiation, which could be related to alterations in PAs and amino acids pools.

The involvement of auxin, abscisic acid (ABA), polyamines (PAs), and proline in adaptation to long-term exposure of woody plants to high levels of heavy metals in soil has received scant attention, even in poplar which is a good candidate for phytoremediation of metal-polluted soils and is regarded as a model for basic research in tree species. Three poplar clones (M1, PE19/66, and B229) were comparatively analyzed in a pot experiment for their responses to 300 mg kg-1 Cu(NO3)2 at morphological, physiological, and biochemical levels. After 4 months, despite the prevalent accumulation of Cu in roots, where the metal reached potentially toxic concentrations, the three clones showed distinct Cu accumulation and translocation capacities, whereas they did not display evident toxicity symptoms or growth inhibition. Several protective mechanisms, namely decreased photosynthetic functionality, enhanced guaiacol peroxidase (GPOD) activity, and accumulation of proline and PAs, were differentially activated in Cu-treated plants in an organ- and clone-specific manner. Overall, a positive relationship between root Cu concentration with GPOD, proline, and PAs was observed. In M1, higher Cu accumulation in roots and leaves compared with other clones was reflected in stimulation of GPOD activity in both organs and in enhanced proline, and PA levels. In PE19/66, these responses were observed only in roots concomitant with high Cu accumulation. Clone B229 accumulated very low amounts of Cu, therefore, these defense responses were attenuated compared with other clones. Enhanced ABA concentrations in response to Cu were observed in PE19/66 and B229; this was likely responsible for stomatal limitation of photosynthesis in PE19/66, whereas in B229 this effect may have been counteracted by increased IAA. Essentially unchanged leaf auxin levels under Cu stress may account for the lack of shoot growth inhibition observed in all three clones; B229 was the only clone that displayed Cu-induced IAA accumulation in roots. Results are discussed in terms of clone-specific adaptive mechanisms to Cu stress in tolerant poplars.

Recent findings in nanomedicine have revealed that carbon nanotubes (CNTs) can be used as potential drug carriers, therapeutic agents and diagnostics tools. Moreover, due to their ability to cross cellular membranes, their nanosize dimension, high surface area and relatively good biocompatibility, CNTs have also been employed as a novel gene delivery vector system. In our previous work, we functionalized CNTs with two polyamine polymers, polyethyleneimine (PEI) and polyamidoamine dendrimer (PAMAM). These compounds have low cytotoxicity, ability to conjugate microRNAs (such as miR-503) and, at the same time, transfect efficiently endothelial cells. The parameters contributing to the good efficiency of transfection that we observed were not investigated in detail. In fact, the diameter and length of CNTs are important parameters to be taken into account when evaluating the effects on drug delivery efficiency. In order to investigate the biophysical and biological contributions of polymer-coated CNTs in delivery of miRNAs to human cells, we decided to investigate three different preparations, characterized by different dimensions and aspect ratios. In particular, we took into account very small CNTs, a suspension of CNTs starting from the commercial product and a 2D material based on CNTs (ie, buckypapers [BPs]) to examine the transfection efficiency of a rigid scaffold. In conclusion, we extensively investigated the biophysical and biological contributions of polyamine-coated CNTs and bidimensional BPs in the delivery of miRNAs to human cells, in order to optimize the transfection efficiency of these compounds to be employed as efficient drug delivery vectors in biomedical applications.

An anion exchange membrane (AEM) was made with a modified polyketone (PK). AEMs of polyamines were prepared in a three-step procedure: (I) PK synthesis using ethylene and carbon monoxide, supported by a Pd catalyst, followed by the introduction of 1,2-diaminopropane to yield the polymeric amines; (II) solvent casting of the modified PK with a low degree of amination; (III) iodomethylation to form the AEM (PK-PDAPm(I)), followed by ion exchange with KOH (PK-PDAPm(OH)). The structure of the modified polyketone was characterized using FT-IR, and UV-vis spectroscopy, demonstrating the successful introduction of amine in the PK. The conductivity of the AEM was studied using broadband electric spectroscopy (BES) in the temperature range from -100 to 120 °C: the highest value of 9 × 10-4 S·cm-1 was reached at 120 °C for the ionic conductivity of PK-PDAPm(I), followed by PK-PDAPm(OH) with values of the same order of magnitude (10-4 S·cm-1). Thermogravimetry showed that the material is thermally stable up to 200 °C.

Introduction: Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments. Objectives: We evaluated the influence of genetic background (high polyamine lines; low methyl jasmonate line; low ethylene line; and isogenic genotypes carrying double transgenic events) and environments (hairy vetch, rye, plastic black mulch and bare soil mulching systems) on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined. Methods: NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics. Results: High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop. Conclusion: This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.

Eucalyptus is a promising species for ecological restoration but plant performances under environmental constraints need to be better investigated. In particular, the toxic effects of metals on this plant speciesare poorly described in the literature. In this work, morpho-physiological and biochemical responses tocadmium were analysed in two eucalypt genotypes (hybrid clones of Eucalyptus camaldulensis × Eucalyptus globulus ssp. bicostata J.B. Kirkp named Velino ex 7 and Viglio ex 358) exposed for 3 weeks to 50 ?M CdSO<inf>4</inf> under hydroponics.The two eucalypt clones showed a different sensitivity to the metal. The growth reduction caused by cadmium was less than 30% in clone Velino and about 50% in clone Viglio. Cadmium mostly accumulated in plant roots and, to a lesser extent, in stem, as highlighted by the low translocation factor (Tf) measured in both clones. Net photosynthesis measurement, chlorophyll fluorescence images, transpiration values and chlorophyll content revealed a cadmium-induced impairment of physiological processes at the leaf level, which was more evident in clone Viglio. Metal binding and antioxidative compound content was differentially affected by cadmium exposure in the two eucalypt clones. Particularly, the content of thiols like cysteine and glutathione, organic acids like oxalate and citrate, and polyamines were markedly modulated in plant organs by metal treatment and highlighted different defence responses between the clones. Cadmium tolerance and accumulation ability of the eucalypt clones were evaluated and the potential of E.camaldulensis for the reclamation of metal polluted-waters is discussed.

Among metals, Ni has been indicated as one of the most dangerous for the environment, and plants exposed to this metal are frequently reported to undergo a severe stress condition. In this work, the tolerance responses to different Ni concentrations at physiological and biochemical levels were evaluated in Amaranthus paniculatus L., a plant species previously characterised for their ability to phytoremove Ni from metal-spiked water. Results indicated a good metal tolerance of this plant species at environmentally relevant Ni concentrations, while clear symptoms of oxidative damages were detected at higher Ni concentrations, both in roots and leaves, by measuring lipid peroxide content. At the photosynthetic level, pigment content determination, chlorophyll fluorescence image analysis and gas-exchange parameter measurements revealed a progressive impairment of the photosynthetic machinery at increasing Ni concentrations in the solution. Regarding biochemical mechanisms involved in antioxidative defence and metal binding, antioxidative enzyme (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX; superoxide dismutase, SOD) activity, polyamine (PA) content, polyamine oxidase (PAO) activity and organic acid (OA) content were differently affected by Ni concentration in the growth solution. A role for GPX, SOD, PAs, and oxalic and citric acid in Ni detoxification is suggested. These results can contribute to elucidate the tolerance mechanisms carried out by plants when facing environmentally relevant Ni concentrations and to identify some traits characterising the physiological and biochemical responses of Amaranthus plants to the presence and bioaccumulation of Ni.

Among metals, Ni has been indicated as one of the most dangerous for the environment, and plants exposed to this metal are frequently reported to undergo a severe stress condition. In this work, the tolerance responses to different Ni concentrations at physiological and biochemical levels were evaluated in Amaranthus paniculatus L., a plant species previously characterised for their ability to phytoremove Ni from metal-spiked water. Results indicated a good metal tolerance of this plant species at environmentally relevant Ni concentrations, while clear symptoms of oxidative damages were detected at higher Ni concentrations, both in roots and leaves, by measuring lipid peroxide content. At the photosynthetic level, pigment content determination, chlorophyll fluorescence image analysis and gas-exchange parameter measurements revealed a progressive impairment of the photosynthetic machinery at increasing Ni concentrations in the solution. Regarding biochemical mechanisms involved in antioxidative defence and metal binding, antioxidative enzyme (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX; superoxide dismutase, SOD) activity, polyamine (PA) content, polyamine oxidase (PAO) activity and organic acid (OA) content were differently affected by Ni concentration in the growth solution. A role for GPX, SOD, PAs, and oxalic and citric acid in Ni detoxification is suggested. These results can contribute to elucidate the tolerance mechanisms carried out by plants when facing environmentally relevant Ni concentrations and to identify some traits characterising the physiological and biochemical responses of Amaranthus plants to the presence and bioaccumulation of Ni.

Polyamines (PA) are believed to protect DNA minimizing the effect of radiation damage either by inducing DNA compaction and aggregation or acting as scavengers of free radicals. Using an in vitro pDNA double strand breakage assay based on gel electrophoretic mobility, we compared the protective capability of PA against ?-radiation with that of compounds generated by the supramolecular self-assembly of nuclear polyamines and phosphates, named Nuclear Aggregates of Polyamines (NAPs). Both unassembled PA and in vitro produced NAPs (ivNAPs) were ineffective in conferring pDNA protection at the sub-mM concentration. Single PA showed an appreciable protective effect only at high (mM) concentrations. However, concentrations of spermine (4+) within a critical range (0.48 - 1 mM) induced pDNA precipitation, an event that was not observed with NAPs-pDNA interaction. We conclude that the interaction of individual PA is ineffective to assure DNA protection, simultaneously preserving the flexibility and charge density of the double strand. Furthermore, data obtained by testing polyamine and ivNAPS with the current radiation-induced DNA damage model support the concept that PA-phosphate aggregates are the only forms through which PA interact with DNA

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches. © 2013 Elsevier GmbH.

Pathoadaptive mutations are evolutionary events leading to the silencing of specific anti-virulence loci. This reshapes the core genome of a novel pathogen, adapts it to the host and boosts its harmful potential. A paradigmatic case is the emergence of Shigella, the causative agent of bacillary dysentery, from its innocuous Escherichia coli ancestor. Here we summarize current views on how pathoadaptation has allowed Shigella to progressively increase its virulence. In this context, modification of the polyamine pattern emerges as a crucial step towards full expression of the virulence program in Shigella. © 2012 Institut Pasteur.

Release of pharmaceuticals in the environment has been emerging as a great concern for ecosystem and human health. Ibuprofen (IBU) represents one of the most widespread pharmaceuticals in surface waters and sediments in spite of the high removal rates occurring in conventional wastewater technologies. To assess the potentiality of phytoremediation in assisting these technologies, the screening of plant species for tolerance and removal ability of pollutants is a very important issue. In this study, the effects of different IBU concentrations on callus cultures of Populus nigra L., a pioneer tree species in the riparian ecosystem, were investigated. Results evidenced a notable tolerance of poplar cells to IBU, especially at high concentrations (IBU 30 mg L-1), which even stimulated growth. At this concentration, the ability to withstand IBU was accompanied by inhibition of lipoxygenase (LOX) activity, reduction of lipid peroxide content and increase of membrane redox activity. Irrespective of initial IBU concentrations, a complete removal of this compound from the growth medium by poplar cells during a subculture occurred. Antioxidative enzyme activities and polyamine content were stimulated by IBU 0.03 mg L-1, while no effect was found in cells exposed to IBU 30 mg L-1, except for a decrease of guaiacol peroxidase (GPX) activity. These findings put in evidence a notable potential of this plant species for the phytoremediation of IBU-contaminated substrates.

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)(2)) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.

The rooting of Vitis (rootstock 140 Ruggeri) woody cuttings, collected in field from mother plants over a five-month period (October 1999-February 2000), was recorded from 0 to 60 days of basal heating. The material was collected directly in field and/or submitted to different treatments: A) cold treatment of the shoots at 2°C in cold chamber from October; b) dipping in water at the bottom end of the cuttings. Both treatments were able to modify rooting time and amount. During basal heating, at 0, 20, 40 and 60 days, rooting percentage was recorded; concomitantly, from the basal end of cuttings, free-polyamines were extracted to evaluate their endogenous levels. Results show that positive variations in rooting, induced by the treatments, are related to increases in free-polyamine levels, suggesting that an increase in the endogenous putrescine could be correlated with pri-mordia formation time. The pattern of spermine variations appears to be similar to that of putrescine, while spermidine does not appear to be correlated to the rooting process. Both spermine and spermidine amounts are present at far lower levels than putrescine.

Spermine oxidase (SMO) is a FAD-containing enzyme involved in animal cell polyamines (PA) homeostasis, selectively active on spermine and producing H(2)O(2), spermidine, and the 3-aminopropanal. In the present study, we have examined the SMO gene expression during the mouse myoblast C2C12 cell differentiation induced with two different stimuli by RT-PCR analysis, polysome-mRNP distribution and enzyme activity. SMO transcript accumulation and enzymatic activity increases during C2C12 cell differentiation and correlates with the decrease of spermine content. Many proteins are highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated post-mitotic myotubes. The SMO gene induction represents a novel and additional marker of C2C12 cell differentiation. The sub-cellular localization of the SMOalpha and SMOmu splice variants is not involved in the differentiation processes. Nuclear localization of only the SMOmu protein was confirmed.

Tobacco (Nicotiana tabacum L. cv. Petit Havana) callus cultures were exposed to UV-C high dose pulse-treatment (254 nm, 50 kJ m-2, 1 h-treatment). After 6, 24 and 48 h from the end of the treatment, calli were cut transversally in two layers and oxidative damage (malondialdehyde [MDA] and hydrogen peroxide), non-enzymatic (radical scavenging antioxidants [RSA] and polyamines) and enzymatic antioxidants (ascorbate peroxidase [APX, EC 1.11.1.11], glutathione reductase [GR, EC 1.6.4.2], catalase [CAT, EC 1.11.1.6] and guaiacol peroxidase [GPX, EC 1.11.1.7]) were evaluated.At each time-point data referred toUV-C treated calli were compared to data of untreated ones (control). Despite of a strong increase of H2O2 content, a slight cellular damage was observed in both upper and lower layers 24 and 48 h after UV-C treatment. An activation first of non-enzymatic antioxidants and then of enzymatic antioxidants was detected in UV-C treated calli. In particular, RSA and putrescine (PUT) accumulated 6 h after UV-C treatment while APX, GR and GPX enzyme activities increased 24 h after UV-C irradiation. Catalase activity did not change. UV-C-induced oxidative stress and antioxidative response were observed also in cell layers not directly exposed to UV irradiation, indicating that a stress signal was transmitted to the whole mass of callus.

Synthesis and characterization of [1+1] macrocyclic and [1+2] macroacyclic compartmental ligands (H2L), containing one N2O2, N3O2, N2O3, N4O2 or O2N2O2 Schiff base site (or the reduced homologues) and one O2O n ( ,4) crown-ether like site, their sodium analogues Na2(L) or Na(HL) nH2O, and the related complexes (Ni, Cu, Mn, Co).