Natural deep eutectic solvents protect RNA from thermal-induced degradation.

RNA is a fundamental nucleic acid for life and it plays important roles in the regulation of gene transcription, post-transcriptional regulation, and epigenetic regulation. Recently, the focus on this nucleic acid has significantly increased due to the development of mRNA vaccines and RNA-based gene therapy protocols. Unfortunately, RNA based products show constrains mainly owing to instability and easy degradability of the RNA molecules. Indeed, unlike the DNA molecule which has a great intrinsic stability, RNA is more prone to degradation and this process is accelerated under thermal treatment. Here we describe a method that involves the use of Natural Deep Eutectic Solvents (NaDES) capable of slowing down RNA degradation process. Our results show that this technology seems suitable for improving the stability of specific RNA molecules particularly susceptible to thermal-induced degradation. Therefore, this technique represents a valuable tool to stabilize RNA molecules used in gene therapy and mRNA vaccines.

Effect of Natural Deep Eutectic Solvents on trans-Resveratrol Photo-Chemical Induced Isomerization and 2,4,6-Trihydroxyphenanthrene Electro-Cyclic Formation.

trans-Resveratrol is a natural bioactive compound with well-recognized health promoting effects. When exposed to UV light, this compound can undergo a photochemically induced trans/cis isomerization and a 6π electrochemical cyclization with the subsequent formation of 2,4,6-trihydroxyphenanthrene (THP). THP is a potentially harmful compound which can exert genotoxic effects. In this work we improved the chromatographic separation and determination of the two resveratrol isomers and of THP by using a non-commercial pentafluorophenyl stationary phase. We assessed the effect of natural deep eutectic solvents (NaDES) as possible photo-protective agents by evaluating cis-resveratrol isomer and THP formation under different UV-light exposure conditions with the aim of enhancing resveratrol photostability and inhibiting THP production. Our results demonstrate a marked photoprotective effect exerted by glycerol-containing NaDES, and in particular by proline/glycerol NaDES, which exerts a strong inhibitory effect on the photochemical isomerization of resveratrol and significantly limits the formation of the toxic derivative THP. Considering the presence of resveratrol in various commercial products, these results are of note in view of the potential genotoxic risk associated with its photochemical degradation products and in view of the need for the development of green, eco-sustainable and biocompatible resveratrol photo-stable formulations.

Green Route for the Isolation and Purification of Hyrdoxytyrosol, Tyrosol, Oleacein and Oleocanthal from Extra Virgin Olive Oil.

Extra virgin olive oil (EVOO) phenols represent a significant part of the intake of antioxidants and bioactive compounds in the Mediterranean diet. In particular, hydroxytyrosol (HTyr), tyrosol (Tyr), and the secoiridoids oleacein and oleocanthal play central roles as anti-inflammatory, neuro-protective and anti-cancer agents. These compounds cannot be easily obtained via chemical synthesis, and their isolation and purification from EVOO is cumbersome. Indeed, both processes involve the use of large volumes of organic solvents, hazardous reagents and several chromatographic steps. In this work we propose a novel optimized procedure for the green extraction, isolation and purification of HTyr, Tyr, oleacein and oleocanthal directly from EVOO, by using a Natural Deep Eutectic Solvent (NaDES) as an extracting phase, coupled with preparative high-performance liquid chromatography. This purification method allows the total recovery of the four components as single pure compounds directly from EVOO, in a rapid, economic and ecologically sustainable way, which utilizes biocompatible reagents and strongly limits the use or generation of hazardous substances.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

Amine oxidase-based biosensors for spermine and spermidine determination.

The present work describes the development and optimization of electrochemical biosensors for specific determination of the biogenic polyamine spermine (Spm) and spermidine (Spmd) whose assessment represents a novel important analytical tool in food analysis and human diagnostics. These biosensors have been prepared using novel engineered enzymes: polyamine oxidase (PAO) endowed with selectivity towards Spm and Spmd and spermine oxidase (SMO) characterized by strict specificity towards Spm. The current design entails biosensors in which the enzymes were entrapped in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), a photocrosslinkable gel, onto an electrode surface. Screen-printed electrodes (SPEs) were used as electrochemical transducers for enzymatically produced hydrogen peroxide, operating at different potential vs Ag/AgCl according to the material of the working electrode (WE): +700 mV for graphite (GP) or -100 mV for Prussian blue (PB)-modified SPE, respectively. Biosensor performances were evaluated by means of flow injection amperometric (FIA) measurements. The modified electrodes showed good sensitivity, long-term stability and reproducibility. Under optimal conditions, the PAO biosensor showed a linear range 0.003-0.3 mM for Spm and 0.01-0.4 mM for Spmd, while with the SMO biosensor, a linear range of 0.004-0.5 mM for Spm has been obtained. The main kinetic parameters apparent Michaelis constant (K M), turnover number (K cat) and steady-state current (I max) were determined. The proposed device was then applied to the determination of biogenic amines in blood samples. The results obtained were in good agreement with those obtained with the GC-MS reference method.

The Glu²¹6/Ser²¹8 pocket is a major determinant of spermine oxidase substrate specificity.

SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N¹-acetylspermine and N¹-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme-substrate complex have identified Glu²¹6-Ser²¹8 as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N¹-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines.

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine.

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from -7.6 to -10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of Ki being 0.10 µM and 0.55 µM for acetylpolyamine oxidase and spermine oxidase, respectively.

Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation.

Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G(1) and G(2) phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N(1)-dimethylthiourea, a hydrogen peroxide (H(2)O(2)) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H(2)O(2) production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H(2)O(2) derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.

The members of Arabidopsis thaliana PAO gene family exhibit distinct tissue- and organ-specific expression pattern during seedling growth and flower development.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis thaliana, five PAOs (AtPAO1-5) are present with cytosolic or peroxisomal localization. Here, we present a detailed study of the expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 during seedling and flower growth and development through analysis of promoter activity in AtPAO::ß-glucuronidase (GUS) transgenic Arabidopsis plants. The results reveal distinct expression patterns for each studied member of the AtPAO gene family. AtPAO1 is mostly expressed in the transition region between the meristematic and the elongation zone of roots and anther tapetum, AtPAO2 in the quiescent center, columella initials and pollen, AtPAO3 in columella, guard cells and pollen, and AtPAO5 in the vascular system of roots and hypocotyls. Furthermore, treatment with the plant hormone abscisic acid induced expression of AtPAO1 in root tip and AtPAO2 in guard cells. These data suggest distinct physiological role(s) for each member of the AtPAO gene family.

Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants.

Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H(2)O(2) in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants.

Clinical utility of a novel colorimetric chair side test for oral malodour.

AIM: The aim of this study was to evaluate the utility of a simple colorimetric chair side test detecting amines in saliva as an adjunct test in the oral malodour diagnosis. MATERIAL AND METHODS: Non-stimulated saliva samples were collected from 100 volunteers with different degrees of oral malodour. The amount of amines detected by the test was estimated clinically (colorimetric test), confirmed semi quantitatively in the laboratory (standard addition method) and consequently compared with (i) the organoleptic score (OLS); (ii) the volatile sulphur compounds (VSCs) levels (OralChroma(™)) and (iii) the amount of amines detected by means of gas chromatography - mass spectroscopy. RESULTS: The chair side test correlated well (Spearman correlation coefficient: 0.46-0.77), with the OLS, the level of VSCs, and the amines determined by using gas chromatography - mass spectroscopy. The results of the new test for patients with and without oral malodour were significantly different (Mann-Whitney U-test, p < 0.0001). The sensitivity, specificity, and positive and negative predictive value of this chair side test were similar to those of the VSCs evaluations. CONCLUSIONS: These results support the "fit for purpose" of the new chair side test as adjunctive diagnostic tool for oral malodour.

Functional diversity inside the Arabidopsis polyamine oxidase gene family.

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. All so far characterized PAOs from monocotyledonous plants, such as the apoplastic maize PAO, oxidize spermine (Spm) and spermidine (Spd) to produce 1,3-diaminopropane, H(2)O(2), and an aminoaldehyde, and are thus considered to be involved in a terminal catabolic pathway. Mammalian PAOs oxidize Spm or Spd (and/or their acetyl derivatives) differently from monocotyledonous PAOs, producing Spd or putrescine, respectively, in addition to H(2)O(2) and an aminoaldehyde, and are therefore involved in a polyamine back-conversion pathway. In Arabidopsis thaliana, five PAOs (AtPAO1-AtPAO5) are present with cytosolic or peroxisomal localization and three of them (the peroxisomal AtPAO2, AtPAO3, and AtPAO4) form a distinct PAO subfamily. Here, a comparative study of the catalytic properties of recombinant AtPAO1, AtPAO2, AtPAO3, and AtPAO4 is presented, which shows that all four enzymes strongly resemble their mammalian counterparts, being able to oxidize the common polyamines Spd and/or Spm through a polyamine back-conversion pathway. The existence of this pathway in Arabidopsis plants is also evidenced in vivo. These enzymes are also able to oxidize the naturally occurring uncommon polyamines norspermine and thermospermine, the latter being involved in important plant developmental processes. Furthermore, data herein reveal some important differences in substrate specificity among the various AtPAOs, which suggest functional diversity inside the AtPAO gene family. These results represent a new starting point for further understanding of the physiological role(s) of the polyamine catabolic pathways in plants.

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.

Characterization and application of a diamine oxidase from Lathyrus sativus as component of an electrochemical biosensor for the determination of biogenic amines in wine and beer.

In this work, we have characterized a diamine oxidase (DAO) from Lathyrus sativus and evaluated its use, for the first time, as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples. Firstly, DAO was electrokinetically characterized free in solution by means of a platinum electrode and then immobilized by using polyazetidine prepolimer on the surface of screen-printed electrodes constituted of two gold working electrodes. The amperometric measurements were carried out by using a flow system at a fixed potential of +600 mV vs the internal silver pseudo reference in phosphate buffer solution (0.1 mol l(-1), pH = 7.4). The analysis of wine and beer samples were performed in flow injection system using the dual channel transducer providing simultaneous detection of sample and blank signal, and the resulting signal (after subtraction of the blank signal) was referred to that of putrescine. The results were compared with those obtained using a modified reference method based on gas chromatography-mass spectrometry analysis on the same samples. The results obtained in the analysis of Italian wines shows the better suitability of DAO-based biosensor in the determination of the biogenic amines (BAs) index expressed as putrescine equivalent in both red and white wines, being less efficient in beer samples where it underestimates by about 50% the BAs content.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm.

BACKGROUND: Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. METHODS: BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. RESULTS: Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. CONCLUSIONS: This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis.

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 degrees C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 degrees C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin.

Functions of amine oxidases in plant development and defence.

Copper amine oxidases and flavin-containing amine oxidases catalyse the oxidative de-amination of polyamines, which are ubiquitous compounds essential for cell growth and proliferation. Far from being only a means of degrading cellular polyamines and, thus, contributing to polyamine homeostasis, amine oxidases participate in important physiological processes through their reaction products. In plants, the production of hydrogen peroxide (H(2)O(2)) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion. As a signal molecule, H(2)O(2) derived from polyamine oxidation mediates cell death, the hypersensitive response and the expression of defence genes. Furthermore, aminoaldehydes and 1,3-diaminopropane from polyamine oxidation are involved in secondary metabolite synthesis and abiotic stress tolerance.

Beneficial effects of a plant histaminase in a rat model of splanchnic artery occlusion and reperfusion.

Splanchnic artery occlusion (SAO) followed by reperfusion causes endothelial injury and inflammation which contribute to the pathophysiology of shock. We investigated the effects of pea seedling (Latyrus cicera) histaminase, known to afford protection against the deleterious effects of cardiac ischemia/reperfusion, given to rats subjected to SAO/reperfusion-induced splanchnic injury. Histaminase (80 IU kg, 15 min before reperfusion) significantly reduced the drop of blood pressure and high mortality rate caused by SAO/reperfusion. Histaminase also reduced histopathological changes, leukocyte infiltration (myeloperoxidase), and expression of endothelial cell adhesion molecules in the ileum. Histaminase counteracted free radical-mediated tissue injury, as judged by a significant decrease in the plasma and tissue levels of peroxidation and nitration products (oxidized rhodamine, malondialdehyde, nitrotyrosine), DNA damage markers (8-hydroxy-2'-deoxyguanosine, poly-adenosine diphosphate-ribosylated DNA) and consumption of tissue antioxidant enzymes (superoxide dismutase). As a result, histaminase led to a reduction of ileal cell apoptosis (caspase 3, terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive cells). These results show that histaminase exerts a clear-cut protective effect in SAO/reperfusion-induced splanchnic injury, likely caused by oxidative catabolism of proinflammatory histamine and antioxidant effects resulting in hindrance of free radical-mediated tissue injury, endothelial dysfunction, and leukocyte recruitment. Thus, histaminase could be used therapeutically in intestinal ischemia.