L-Aminoguanidine Induces Imbalance of ROS/RNS Homeostasis and Polyamine Catabolism of Tomato Roots after Short-Term Salt Exposure.

Polyamine (PA) catabolism mediated by amine oxidases is an important process involved in fine-tuning PA homeostasis and related mechanisms during salt stress. The significance of these amine oxidases in short-term responses to salt stress is, however, not well understood. In the present study, the effects of L-aminoguanidine (AG) on tomato roots treated with short-term salt stress induced by NaCl were studied. AG is usually used as a copper amine oxidase (CuAO or DAO) inhibitor. In our study, other alterations of PA catabolism, such as reduced polyamine oxidase (PAO), were also observed in AG-treated plants. Salt stress led to an increase in the reactive oxygen and nitrogen species in tomato root apices, evidenced by in situ fluorescent staining and an increase in free PA levels. Such alterations were alleviated by AG treatment, showing the possible antioxidant effect of AG in tomato roots exposed to salt stress. PA catabolic enzyme activities decreased, while the imbalance of hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) concentrations displayed a dependence on stress intensity. These changes suggest that AG-mediated inhibition could dramatically rearrange PA catabolism and related reactive species backgrounds, especially the NO-related mechanisms. More studies are, however, needed to decipher the precise mode of action of AG in plants exposed to stress treatments.

Serial femtosecond X-ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase.

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers (XFELs) has made it possible to address these issues. In particular, mix-and-inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid-jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Šresolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single-turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid-jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time-resolved MISC to study enzymic reaction mechanisms under anaerobic conditions.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. ßAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). ßAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Effects of Chemical Structures Interacting with Amine Oxidases on Glucose, Lipid and Hydrogen Peroxide Handling by Human Adipocytes.

Benzylamine is a natural molecule present in food and edible plants, capable of activating hexose uptake and inhibiting lipolysis in human fat cells. These effects are dependent on its oxidation by amine oxidases present in adipocytes, and on the subsequent hydrogen peroxide production, known to exhibit insulin-like actions. Virtually, other substrates interacting with such hydrogen peroxide-releasing enzymes potentially can modulate lipid accumulation in adipose tissue. Inhibition of such enzymes has also been reported to influence lipid deposition. We have therefore studied in human adipocytes the lipolytic and lipogenic activities of pharmacological entities designed to interact with amine oxidases highly expressed in this cell type: the semicarbazide-sensitive amine oxidase (SSAO also known as PrAO or VAP-1) and the monoamine oxidases (MAO). The results showed that SZV-2016 and SZV-2017 behaved as better substrates than benzylamine, releasing hydrogen peroxide once oxidized, and reproduced or even exceeded its insulin-like metabolic effects in fat cells. Additionally, several novel SSAO inhibitors, such as SZV-2007 and SZV-1398, have been evidenced and shown to inhibit benzylamine metabolic actions. Taken as a whole, our findings reinforce the list of molecules that influence the regulation of triacylglycerol assembly/breakdown, at least in vitro in human adipocytes. The novel compounds deserve deeper investigation of their mechanisms of interaction with SSAO or MAO, and constitute potential candidates for therapeutic use in obesity and diabetes.

Hyperglycemia and reduced adiposity of streptozotocin-induced diabetic mice are not alleviated by oral benzylamine supplementation.

BACKGROUND: Benzylamine (Bza) oral administration delays the onset of hyperglycemia in insulin-resistant db -/- mice; a genetic model of obesity and type 2 diabetes. AIM: To extend the antihyperglycemic properties of oral benzylamine to a model of insulin-deficient type 1 diabetes. METHODS: Male Swiss mice were rendered diabetic by streptozotocin treatment (STZ) and divided in two groups: one received 0.5% Bza as drinking solution for 24 d (STZ Bza-drinking) while the other was drinking water ad libitum. Similar groups were constituted in age-matched, nondiabetic mice. Food intake, liquid intake, body weight gain and nonfasting blood glucose levels were followed during treatment. At the end of treatment, fasted glycemia, liver and white adipose tissue (WAT) mass were measured, while glucose uptake assays were performed in adipocytes. RESULTS: STZ diabetic mice presented typical features of insulin-deficient diabetes: reduced body mass and increased blood glucose levels. These altered parameters were not normalized in the Bza-drinking group in spite of restored food and water intake. Bza consumption could not reverse the severe fat depot atrophy of STZ diabetic mice. In the nondiabetic mice, no difference was found between control and Bza-drinking mice for any parameter. In isolated adipocytes, hexose uptake was partially activated by 0.1 mmol/L Bza in a manner that was obliterated in vitro by the amine oxidase inhibitor phenelzine and that remained unchanged after Bza supplementation. Oxidation of 0.1 mmol/L Bza in WAT was lower in STZ diabetic than in normoglycemic mice. CONCLUSION: Bza supplementation could not normalize the altered glucose handling of STZ diabetic mice with severe WAT atrophy. Consequently, its antidiabetic potential in obese and diabetic rodents does not apply to lipoatrophic type 1 diabetic mice.

Re-evaluation of protein neutron crystallography with and without X-ray/neutron joint refinement.

Protein neutron crystallography is a powerful technique to determine the positions of H atoms, providing crucial biochemical information such as the protonation states of catalytic groups and the geometry of hydrogen bonds. Recently, the crystal structure of a bacterial copper amine oxidase was determined by joint refinement using X-ray and neutron diffraction data sets at resolutions of 1.14 and 1.72 Å, respectively [Murakawa et al. (2020 ▸). Proc. Natl Acad. Sci. USA, 117, 10818-10824]. While joint refinement is effective for the determination of the accurate positions of heavy atoms on the basis of the electron density, the structural information on light atoms (hydrogen and deuterium) derived from the neutron diffraction data might be affected by the X-ray data. To unravel the information included in the neutron diffraction data, the structure determination was conducted again using only the neutron diffraction data at 1.72 Šresolution and the results were compared with those obtained in the previous study. Most H and D atoms were identified at essentially the same positions in both the neutron-only and the X-ray/neutron joint refinements. Nevertheless, neutron-only refinement was found to be less effective than joint refinement in providing very accurate heavy-atom coordinates that lead to significant improvement of the neutron scattering length density map, especially for the active-site cofactor. Consequently, it was confirmed that X-ray/neutron joint refinement is crucial for determination of the real chemical structure of the catalytic site of the enzyme.

Increased monoamine oxidase activity and imidazoline binding sites in insulin-resistant adipocytes from obese Zucker rats.

BACKGROUND: Despite overt insulin resistance, adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids. AIM: To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide, since it is produced by monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in adipocytes. METHODS: 3H-2-deoxyglucose uptake (2-DG) was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine. 14C-tyramine oxidation and binding of imidazolinic radioligands [3H-Idazoxan, 3H-(2-benzofuranyl)-2-imidazoline] were studied in adipocytes, the liver, and muscle. The influence of in vivo administration of tyramine + vanadium on glucose handling was assessed in lean and obese rats. RESULTS: 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats, when compared to their lean littermates. Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited, while MAO was increased and SSAO decreased. These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat, when compared to the lean. In vitro, tyramine precluded the binding to I2 sites, while in vivo, its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese rats. CONCLUSION: The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number. However, probably as a consequence of SSAO down-regulation, the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes. The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

High doses of catecholamines activate glucose transport in human adipocytes independently from adrenoceptor stimulation or vanadium addition.

BACKGROUND: When combined with vanadium salts, catecholamines strongly activate glucose uptake in rat and mouse adipocytes. AIM: To test whether catecholamines activate glucose transport in human adipocytes. METHODS: The uptake of 2-deoxyglucose (2-DG) was measured in adipocytes isolated from pieces of abdominal subcutaneous tissue removed from women undergoing reconstructive surgery. Pharmacological approaches with amine oxidase inhibitors, adrenoreceptor agonists and antioxidants were performed to unravel the mechanisms of action of noradrenaline or adrenaline (also named epinephrine). RESULTS: In human adipocytes, 45-min incubation with 100 µmol/L adrenaline or noradrenaline activated 2-DG uptake up to more than one-third of the maximal response to insulin. This stimulation was not reproduced with millimolar doses of dopamine or serotonin and was not enhanced by addition of vanadate to the incubation medium. Among various natural amines and adrenergic agonists tested, no other molecule was more efficient than adrenaline and noradrenaline in stimulating 2-DG uptake. The effect of the catecholamines was not impaired by pargyline and semicarbazide, contrarily to that of benzylamine or methylamine, which are recognized substrates of semicarbazide-sensitive amine oxidase. Hydrogen peroxide at 1 mmol/L activated hexose uptake but not pyrocatechol or benzoquinone, and only the former was potentiated by vanadate. Catalase and the phosphoinositide 3-kinase inhibitor wortmannin inhibited adrenaline-induced activation of 2-DG uptake. CONCLUSION: High doses of catecholamines exert insulin-like actions on glucose transport in human adipocytes. At submillimolar doses, vanadium did not enhance this catecholamine activation of glucose transport. Consequently, this dismantles our previous suggestion to combine the metal ion with catecholamines to improve the benefit/risk ratio of vanadium-based antidiabetic approaches.

A New Player in Jasmonate-Mediated Stomatal Closure: The Arabidopsis thaliana Copper Amine Oxidase ß.

Plant defence responses to adverse environmental conditions include different stress signalling, allowing plant acclimation and survival. Among these responses one of the most common, immediate, and effective is the modulation of the stomatal aperture, which integrates different transduction pathways involving hydrogen peroxide (H2O2), calcium (Ca2+), nitric oxide (NO), phytohormones and other signalling components. The Arabidopsis thaliana copper amine oxidases ß (AtCuAOß) encodes an apoplastic CuAO expressed in guard cells and root protoxylem tissues which oxidizes polyamines to aminoaldehydes with the production of H2O2 and ammonia. Here, its role in stomatal closure, signalled by the wound-associated phytohormone methyl-jasmonate (MeJA) was explored by pharmacological and genetic approaches. Obtained data show that AtCuAOß tissue-specific expression is induced by MeJA, especially in stomata guard cells. Interestingly, two Atcuaoß T-DNA insertional mutants are unresponsive to this hormone, showing a compromised MeJA-mediated stomatal closure compared to the wild-type (WT) plants. Coherently, Atcuaoß mutants also show compromised H2O2-production in guard cells upon MeJA treatment. Furthermore, the H2O2 scavenger N,N1-dimethylthiourea (DMTU) and the CuAO-specific inhibitor 2-bromoethylamine (2-BrEtA) both reversed the MeJA-induced stomatal closure and the H2O2 production in WT plants. Our data suggest that AtCuAOß is involved in the H2O2 production implicated in MeJA-induced stomatal closure.

Novel Facet of an Old Dietary Molecule? Direct Influence of Caffeine on Glucose and Biogenic Amine Handling by Human Adipocytes.

Caffeine is a plant alkaloid present in food and beverages consumed worldwide. It has high lipid solubility with recognized actions in the central nervous system and in peripheral tissues, notably the adipose depots. However, the literature is scant regarding caffeine's influence on adipocyte functions other than lipolysis, such as glucose incorporation into lipids (lipogenesis) and amine oxidation. The objective of this study was to explore the direct effects of caffeine and of isobutylmethylxanthine (IBMX) on these adipocyte functions. Glucose transport into fat cells freshly isolated from mice, rats, or humans was monitored by determining [3H]-2-deoxyglucose (2-DG) uptake, while the incorporation of radiolabeled glucose into cell lipids was used as an index of lipogenic activity. Oxidation of benzylamine by primary amine oxidase (PrAO) was inhibited by increasing doses of caffeine in human adipose tissue preparations with an inhibition constant (Ki) in the millimolar range. Caffeine inhibited basal and insulin-stimulated glucose transport as well as lipogenesis in rodent adipose cells. The antilipogenic action of caffeine was also observed in adipocytes from mice genetically invalidated for PrAO activity, indicating that PrAO activity was not required for lipogenesis inhibition. These caffeine inhibitory properties were extended to human adipocytes: relative to basal 2-DG uptake, set at 1.0 ± 0.2 for 6 individuals, 0.1 mM caffeine tended to reduce uptake to 0.83 ± 0.08. Insulin increased uptake by 3.86 ± 1.11 fold when tested alone at 100 nM, and by 3.21 ± 0.80 when combined with caffeine. Our results reinforce the recommendation of caffeine's potential in the treatment or prevention of obesity complications.

Plant Copper Amine Oxidases: Key Players in Hormone Signaling Leading to Stress-Induced Phenotypic Plasticity.

Polyamines are ubiquitous, low-molecular-weight aliphatic compounds, present in living organisms and essential for cell growth and differentiation. Copper amine oxidases (CuAOs) oxidize polyamines to aminoaldehydes releasing ammonium and hydrogen peroxide, which participates in the complex network of reactive oxygen species acting as signaling molecules involved in responses to biotic and abiotic stresses. CuAOs have been identified and characterized in different plant species, but the most extensive study on a CuAO gene family has been carried out in Arabidopsis thaliana. Growing attention has been devoted in the last years to the investigation of the CuAO expression pattern during development and in response to an array of stress and stress-related hormones, events in which recent studies have highlighted CuAOs to play a key role by modulation of a multilevel phenotypic plasticity expression. In this review, the attention will be focused on the involvement of different AtCuAOs in the IAA/JA/ABA signal transduction pathways which mediate stress-induced phenotypic plasticity events.

Novel starter cultures Virgibacillus spp. selected from grasshopper sub shrimp paste to inhibit biogenic amines accumulation.

Controlling the content of biogenic amines (BAs) is critical to guarantee the safety of fermented aquatic products. The degradation characteristics and application potential of amine-negative starter cultures (Virgibacillus halodenitrificans CGMCC 1.18601: G25, Virgibacillus pantothenticus CGMCC 1.18602: G38) screened from grasshopper sub shrimp paste (Gssp) were studied. The enzymes of the two strains G25 and G38 that degrade BAs were amine oxidases (AOs) located on their respective cell membranes. The conditions that promoted the AO activity of Virgibacillus spp. were NaCl concentrations 5-10%, temperature 37 °C, pH 7.0 and ethanol concentrations 0-2%. Safety assessments (antibiotic susceptibility, biofilm activity and hemolytic activity) indicated that Virgibacillus spp. do not present a risk to human health, and this isolate can be confidently recommended as safe starter cultures for the food industry. Then, the two strains were cultured separately as starters and applied to the Gssp to analyze their influence on the flavor and quality of the product. As far as the bad flavors in Gssp such as sulfur-organic and sulf-chlor were concerned, the response values in the starter groups by G25 and G38 were significantly reduced by 39% and 65%, respectively. For the ability of strains to degrade BAs in Gssp, G25 degraded 11.1% of histamine, 11.3% of tyramine, 15.5% of putrescine and 4.1% of cadaverine; G38 significantly degraded 10.1% of histamine, 21.8% of tyramine, 18.1% of putrescine and 5.0% of cadaverine. These results indicated that the selected species could be used as starter cultures for the control of BA accumulation and degradation in Gssp.

Obesity of mice lacking VAP-1/SSAO by Aoc3 gene deletion is reproduced in mice expressing a mutated vascular adhesion protein-1 (VAP-1) devoid of amine oxidase activity.

The product of Aoc3 gene is known as vascular adhesion protein-1 (VAP-1), a glycoprotein contributing to leukocyte extravasation and exhibiting semicarbazide-sensitive amine oxidase activity (SSAO). Regarding the immune functions of VAP-1/SSAO, it is known that mice bearing Aoc3 gene knock-out (AOC3KO) exhibit defects in leukocyte migration similar to those of mice expressing a mutated VAP-1 lacking functional SSAO activity (knock-in, AOC3KI). However, it has not been reported whether these models differ regarding other disturbances. Thus, we further compared endocrine-metabolic phenotypes of AOC3KO and AOC3KI mice to their respective control. Special attention was paid on adiposity, glucose and lipid handling, since VAP-1/SSAO is highly expressed in adipose tissue (AT). In both mouse lines, no tissue SSAO activity was found, while Aoc3 mRNA was absent in AOC3KO only. Although food consumption was unchanged, both AOC3KO and AOC3KI mice were heavier and fatter than their respective controls. Other alterations commonly found in adipocytes from both lines were loss of benzylamine insulin-like action with unchanged insulin lipogenic responsiveness and adiponectin expression. A similar downregulation of inflammatory markers (CD45, IL6) was found in AT. Glucose handling and liver mass remained unchanged, while circulating lipid profile was distinctly altered, with increased cholesterol in AOC3KO only. These results suggest that the lack of oxidase activity found in AOC3KI is sufficient to reproduce the metabolic disturbances observed in AOC3KO mice, save those related with cholesterol transport. Modulation of SSAO activity therefore constitutes a potential target for the treatment of cardiometabolic diseases, especially obesity when complicated by low-grade inflammation.

Mutation of Arabidopsis Copper-Containing Amine Oxidase Gene AtCuAOδ Alters Polyamines, Reduces Gibberellin Content and Affects Development.

Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA1, GA7, GA8, GA9, and GA20 was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA3 treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.

Glucosinolate catabolism during postharvest drying determines the ratio of bioactive macamides to deaminated benzenoids in Lepidium meyenii (maca) root flour.

Postharvest processing of maca (Lepidium meyenii Walp., Brassicaceae), a traditional high-altitude Andean root crop, involves slow field drying prior to milling into flour. The progressive tissue dehydration and release of hydrolytic enzymes and substrates from cellular compartments results in the slow accumulation of free monosaccharides, fatty acids and amino acids. A more complex, and faster, kinetic profile is that of glucosinolate breakdown. A number of reactive transient and stable accumulation products are generated during drying, some of which have noteworthy bioactive properties. Among these are macamides, inhibitors of endocannabinoid neurotransmitter degradation in mammalian nervous systems. They result from the condensation of benzyl amine, a glucosinolate hydrolysis product, with free fatty acids released from lipid hydrolysis. Recent research has focused on developing drying processes under controlled conditions that can modulate the biochemistry of glucosinolate hydrolysis to optimize the content of bioactive compounds in the root flour. Low temperature (35 °C) oven-drying of shredded maca roots under controlled air flow generates benzyl amine as primary accumulation product, accounting for up to 94% of hydrolyzed glucosinolate in the flour. Kinetic evidence suggests that both deaminated benzenoids and macamides are allocated from the benzylamine pool through amine oxidase activity or condensation with free fatty acids, accounting for the remaining hydrolyzed glucosinolate (<5%). These activities determine the allocation to either one of these pathways. Later stages of dehydration result in shifts in the molar ratios of deaminated benzenoids, the accumulation of benzoic acid esters and benzyl alcohol. We propose that these are the result of changes in the rates of the reductive and oxidative half-reactions of endogenous aldehyde dehydrogenases. It is the ratio of benzylamine deamination to amide formation that determines the eventual yields of macamides in relation to benzenoids and their esters in maca flour.

Human Copper-Containing Amine Oxidases in Drug Design and Development.

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

Leaf-Wounding Long-Distance Signaling Targets AtCuAOß Leading to Root Phenotypic Plasticity.

The Arabidopsis gene AtCuAOß (At4g14940) encodes an apoplastic copper amine oxidase (CuAO) highly expressed in guard cells of leaves and flowers and in root vascular tissues, especially in protoxylem and metaxylem precursors, where its expression is strongly induced by the wound signal methyl jasmonate (MeJA). The hydrogen peroxide (H2O2) derived by the AtCuAOß-driven oxidation of the substrate putrescine (Put), mediates the MeJA-induced early root protoxylem differentiation. Considering that early root protoxylem maturation was also induced by both exogenous Put and leaf wounding through a signaling pathway involving H2O2, in the present study we investigated the role of AtCuAOß in the leaf wounding-induced early protoxylem differentiation in combination with Put treatment. Quantitative and tissue specific analysis of AtCuAOß gene expression by RT-qPCR and promoter::green fluorescent protein-ß-glucuronidase fusion analysis revealed that wounding of the cotiledonary leaf induced AtCuAOß gene expression which was particularly evident in root vascular tissues. AtCuAOß loss-of-function mutants were unresponsive to the injury, not showing altered phenotype upon wounding in comparison to wild type seedlings. Exogenous Put and wounding did not show synergy in inducing early root protoxylem maturation, suggesting their involvement in a shared signaling pathway.

Vanadium-dependent activation of glucose transport in adipocytes by catecholamines is not mediated via adrenoceptor stimulation or monoamine oxidase activity.

BACKGROUND: Benzylamine and methylamine activate glucose uptake in adipocytes. For tyramine, this effect has even been extended to cardiomyocytes. AIM: To investigate the effects of catecholamines and other amines on glucose uptake. METHODS: A screening compared 25 biogenic amines on 2-deoxyglucose (2-DG) uptake activation in rat adipocytes. Pharmacological approaches and transgenic mouse models were then used to decipher the mode of action of several hits. RESULTS: In rat adipocytes, insulin stimulation of 2-DG uptake was reproduced with catecholamines. 100 µmol/L or 1 mmol/L adrenaline, noradrenaline, dopamine and deoxyepinephrine, maximally activated hexose transport only when sodium orthovanadate was added at 100 µmol/L. Such activation was similar to that already reported for benzylamine, methylamine and tyramine, well-recognized substrates of semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase (MAO). Several, but not all, tested agonists of ß-adrenoreceptors (ß-ARs) also activated glucose transport while α-AR agonists were inactive. Lack of blockade by α- and ß-AR antagonists indicated that catecholamine-induced 2-DG uptake was not mediated by AR stimulation. Adipocytes from mice lacking ß1-, ß2- and ß3-ARs (triple KO) also responded to millimolar doses of adrenaline or noradrenaline by activating hexose transport in the presence of 100 µmol/L vanadate. The MAO blocker pargyline, and SSAO inhibitors did not block the effects of adrenaline or noradrenaline plus vanadate, which were blunted by antioxidants. CONCLUSION: Catecholamines exert unexpected insulin-like actions in adipocytes when combined with vanadium. For limiting insulin resistance by activating glucose consumption at least in fat stores, we propose that catecholamine derivatives combined with vanadium can generate novel complexes that may have low toxicity and promising anti-diabetic properties.

Developmental, hormone- and stress-modulated expression profiles of four members of the Arabidopsis copper-amine oxidase gene family.

Copper-containing amine oxidases (CuAOs) catalyze polyamines (PAs) terminal oxidation producing ammonium, an aminoaldehyde and hydrogen peroxide (H2O2). Plant CuAOs are induced by stress-related hormones, methyl-jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA). In the Arabidopsis genome, eight genes encoding CuAOs have been identified. Here, a comprehensive investigation of the expression pattern of four genes encoding AtCuAOs from the α and γ phylogenetic subfamilies, the two peroxisomal AtCuAOα2 (At1g31690) and AtCuAOα3 (At1g31710) and the two apoplastic AtCuAOγ1 (At1g62810) and AtCuAOγ2 (At3g43670), has been carried out by RT-qPCR and promoter::green fluorescent protein-ß-glucuronidase fusion (GFP-GUS). Expression in hydathodes of new emerging leaves (AtCuAOγ1 and AtCuAOγ2) and/or cotyledons (AtCuAOα2, AtCuAOγ1 and AtCuAOγ2) as well as in vascular tissues of new emerging leaves and in cortical root cells at the division/elongation transition zone (AtCuAOγ1), columella cells (AtCuAOγ2) or hypocotyl and root (AtCuAOα3) was identified. Quantitative and tissue-specific gene expression analysis performed by RT-qPCR and GUS-staining in 5- and 7-day-old seedlings under stress conditions or after treatments with hormones or PAs, revealed that all four AtCuAOs were induced during dehydration recovery, wounding, treatment with indoleacetic acid (IAA) and putrescine (Put). AtCuAOα2, AtCuAOα3, AtCuAOγ1 and AtCuAOγ2 expression in vascular tissues and hydathodes involved in water supply and/or loss, along with a dehydration-recovery dependent gene expression, would suggest a role in water balance homeostasis. Moreover, occurrence in zones where an auxin maximum has been observed along with an IAA-induced alteration of expression profiles, support a role in tissue maturation and xylem differentiation events.

The Copper Amine Oxidase AtCuAOδ Participates in Abscisic Acid-Induced Stomatal Closure in Arabidopsis.

Plant copper amine oxidases (CuAOs) are involved in wound healing, defense against pathogens, methyl-jasmonate-induced protoxylem differentiation, and abscisic acid (ABA)-induced stomatal closure. In the present study, we investigated the role of the Arabidopsis thaliana CuAOδ (AtCuAOδ; At4g12290) in the ABA-mediated stomatal closure by genetic and pharmacological approaches. Obtained data show that AtCuAOδ is up-regulated by ABA and that two Atcuaoδ T-DNA insertional mutants are less responsive to this hormone, showing reduced ABA-mediated stomatal closure and H2O2 accumulation in guard cells as compared to the wild-type (WT) plants. Furthermore, CuAO inhibitors, as well as the hydrogen peroxide (H2O2) scavenger N,N1-dimethylthiourea, reversed most of the ABA-induced stomatal closure in WT plants. Consistently, AtCuAOδ over-expressing transgenic plants display a constitutively increased stomatal closure and increased H2O2 production compared to WT plants. Our data suggest that AtCuAOδ is involved in the H2O2 production related to ABA-induced stomatal closure.