Search

Objectives. Biogenic polyamines are widely present in nature. They are characteristic of both protozoan cells and multicellular organisms. These compounds have a wide range of biological functions and are necessary for normal growth and development of cells. Violation of polyamine homeostasis can cause significant abnormalities in cell functioning, provoking various pathological processes, including oncological and neuropsychiatric diseases. The impact on the “polyamine pathway” is an attractive basis for the creation of many pharmacological agents with a diverse spectrum of action. The purpose of this review is to summarize the results of the studies devoted to understanding the biological activity of compounds of the polyamine series, comparing their biological action with action on certain molecular targets. Due to the structural diversity of this group of substances, it is impossible to fully reflect the currently available data in one review. Therefore, in this work, the main attention is paid to the derivatives, acyclic saturated polyamines.Results. The following aspects are considered: biological functionality, biosynthesis and catabolism, cell transport, and localization of biogenic polyamines in the living systems. Structural analogs and derivatives of biogenic polyamines with antitumor, neuroprotective, antiarrhythmic, antiparasitic, antibacterial, and other biological activities are represented; the relationship between biological activity and the target of exposure is reflected. It was found that the nature of the substituent, the number of cationic centers, and the length of the polyamine chain have a great influence on the nature of the effect.Conclusions. At present, the use of polyamine structures is restrained by cytotoxicity and nonspecific toxic effects on the central nervous system. Further research in the field of biochemistry, cell transport, and a deeper understanding of receptor interaction mechanisms will help making polyamines as the basis for potential drug formulation.

This study reveals an uncovered mechanism for the regulation of polyamine homeostasis through protein arginyl citrullination of antizyme (AZ), a natural inhibitor of ornithine decarboxylase (ODC). ODC is critical for the cellular production of polyamines. AZ binds to ODC dimers and promotes the degradation of ODC via the 26S proteasome. This study demonstrates the protein citrullination of AZ catalyzed by peptidylarginine deiminase type 4 (PAD4) both in vitro and in cells. Upon PAD4 activation, the AZ protein was citrullinated and accumulated, leading to higher levels of ODC proteins in the cell. In the PAD4-overexpressing and activating cells, the levels of ODC enzyme activity and the product putrescine increased with the level of citrullinated AZ proteins and PAD4 activity. Suppressing cellular PAD4 activity reduces the cellular levels of ODC and downregulates cellular polyamines. Furthermore, citrullination of AZ in the C-terminus attenuates AZ function in the inhibition, binding, and degradation of ODC. This paper provides evidence to illustrate that PAD4-mediated AZ citrullination upregulates cellular ODC and polyamines by retarding ODC degradation, thus interfering with the homeostasis of cellular polyamines, which may be an important pathway regulating AZ functions that is relevant to cancer biology.

Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in M-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N1-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met2-Spm) has, under standard assay conditions, an IC50 value of 169 M towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.

Polyamines spermidine and spermine, and their diamine precursor putrescine, are essential for normal cellular functions in both pro- and eukaryotes. Cellular polyamine levels are regulated by biosynthesis, degradation and transport. Transport of dietary and luminal bacterial polyamines in gastrointestinal (GI) tissues plays a significant role in tissue polyamine homeostasis. We have reported that caveolin-1 play an inhibitory role in polyamine uptake in GI tissues. We investigated the mechanism of caveolin-1-regulated polyamine transport. We found that glutathione S-transferase Π(GSTΠ) was secreted from caveolin-1 knockdown cells and stimulated spermidine transport in human colon-derived HCT116 cells. GSTΠ secreted in the medium increased S-glutathionylated protein level in the plasma membrane fraction. Proteomic analysis revealed that actin was S-glutathionylated by GSTΠ. Immunofluorescence microscopy demonstrated that actin filaments around plasma membrane were S-glutathionylated in caveolin-1 knockdown cells. Inhibition of actin remodeling by jasplakinolide caused a decrease in polyamine uptake activity. These data support a model in which caveolin-1 negatively regulates polyamine uptake by inhibiting GSTΠ secretion, which stimulates actin remodeling and endocytosis.

Eukaryotic translation initiation factor 5A2 (EIF5A2) is an essential protein closely related to cellular polyamine homeostasis. It is the only cellular protein that contains the unusual amino acid hypusine [Nɛ-(4-amino-2-hydroxybutyl) lysine] which is necessary for the biochemical activity of EIF5A2 and cellular proliferation. In this study, we aim to characterize chicken EIF5A2 and explore its potential effects on skeletal muscle cell proliferation and differentiation. Here, we found that chicken EIF5A2 has four variant transcripts. The single nucleotide polymorphisms (SNPs) in the 5′-flanking region of EIF5A2 were significantly associated with the chicken growth and fatness traits. Expression pattern of EIF5A2 in skeletal muscle revealed that it was highly expressed at an early embryonic age and then decreased sharply after d 15 of the embryonic age. The luciferase reporter assays confirmed that EIF5A2 was directly targeted by miRNA-223. EIF5A2 was negatively regulated by miR-223 in myoblast. Both 5-Ethynyl-2′-deoxyuridine staining assays and flow cytometry analyses of the cell cycle showed that down-regulation of EIF5A2 could markedly inhibit myoblast proliferation. We found that down-regulation of EIF5A2 could promote myoblast differentiation through promoting the expression of Myogenin (MYOG). MYOG is a muscle-specific transcription factor involved in myogenesis. Altogether, the results indicated that EIF5A2 is an important candidate gene in chicken growth, and down-regulation of EIF5A2 could suppress myoblast proliferation and promote myoblast differentiation by targeting miR-223.

The objective of this study was to investigate the effects of exogenous glutathione (GSH) on the ion balance and micro-distribution, polyamine metabolism (PA) levels, activity of key enzymes, expression of PA synthesis and metabolism genes in tomato (Solanum lycopersicum L. cv. Zhongshu No. 4) seedlings under salt (NaCl) stress (100 mM). we found that NaCl stress caused damage to tomato, including seedling growth inhibition, imbalanced intracellular ion homeostasis caused by the accumulation of Na+ and Cl− in roots and leaves, and reduced transport capacity of K+, Ca2+, and Mg2+ from roots to leaves. Salt stress also led to a significant increase in polyamine levels in leaves. BSO (L-Buthionine-sulfoximine, an inhibitor of key GSH synthesis enzyme gamma- glutamylcysteine synthetase) application under salt stress exacerbated growth inhibition and ion imbalance in tomato seedlings and induced further accumulation of polyamine. However, the application of exogenous GSH reduced the Na+ and Cl- content of roots and leaves of plants under salt stress and NB (NaCl + BSO) treatments, regulated ion homeostasis and microdomain distribution, inhibited the selective transport of Na+, and promoted the selective transport of K+ and Ca2+ from root to leaf, thus alleviating the ion imbalance and poisoning caused by salt stress. In addition, exogenous GSH also down-regulated the activities and transcriptional levels of the PA synthesis- and degradation-related key enzymes ADC (arginine decarboxylase), SAMDC (S-adenosylmethionine decarboxylase), and DAO (diamine oxidase) under salt stress and NB treatments, and up-regulated the activities and the transcriptional levels of PAO (PA oxidase) and ODC (ornithine decarboxylase), thereby reducing the level of PAs and promoting the transformation of polyamines between different morphologies. These findings suggest that exogenous GSH applied to salt-stressed tomato confers salinity tolerance in tomato seedlings by modulating ion absorption, microdomain distribution, and the ion selective transport ratio and by regulating polyamine synthesis and metabolism to maintain cellular ions and polyamine homeostasis. This is the first study to provide evidence of the interaction between GSH and PAs in plant adaptive responses to salinity.

‘Empire’ apples were stored at 0 °C or 3 °C with 0.03 or 5 kPa CO2 and 2.5 kPa O2 for 16 wk. Pools of free, soluble conjugated and bound putrescine, spermidine, and spermine fluctuated in a dynamic manner in response to both temperature and CO2. These results suggest that putrescine homeostasis is under different regulatory control than spermidine/spermine homeostasis.

Polyamines are essential metabolites found in all organisms. Intracellular polyamine levels are tightly maintained by biosynthesis, degradation, uptake and excretion processes that involve regulatory mechanisms – such as the antizyme inhibitory protein – that are conserved across the kingdoms of life, indicating that polyamine levels are critical to cell function. Nonetheless, the biochemical roles of polyamines and their involvement in numerous fundamental cellular processes including aging, cell cycle progression and growth only become apparent when polyamine homeostasis is perturbed. Thus, while polyamines are present in most cells and essential for cell growth, their biochemical functions are largely enigmatic. Studies in fungi have contributed to our basic understanding of polyamines, and might continue to bridge knowledge gaps regarding polyamine metabolism and cell function. Moreover, when considering the impact of fungi – directly or indirectly, for good or for ill – on human society, closing gaps in our understanding of polyamine functions in fungal physiology is an important goal in itself that might lead to the discovery of new targets for enhancing beneficial fungal interactions and diminishing those detrimental to crop and human health. To facilitate progress towards this prospect, here we appraise what is known about polyamine metabolism in fungi, how prevalent polyamines impact fungal physiology and metabolism, and how the levels of each polyamine are maintained in the fungal cell – thus pointing to how they might be perturbed.

Polyamines (PAs) are indispensable polycations ubiquitous to all living cells. Among their many critical functions, PAs contribute to the oxidative balance of the cell. Beginning with studies by the Tabor laboratory in bacteria and yeast, the requirement for PAs as protectors against oxygen radical–mediated damage has been well established in many organisms, including mammals. However, PAs also serve as substrates for oxidation reactions that produce hydrogen peroxide (H(2)O(2)) both intra- and extracellularly. As intracellular concentrations of PAs can reach millimolar concentrations, the H(2)O(2) amounts produced through their catabolism, coupled with a reduction in protective PAs, are sufficient to cause the oxidative damage associated with many pathologies, including cancer. Thus, the maintenance of intracellular polyamine homeostasis may ultimately contribute to the maintenance of oxidative homeostasis. Again, pioneering studies by Tabor and colleagues led the way in first identifying spermine oxidase in Saccharomyces cerevisiae. They also first purified the extracellular bovine serum amine oxidase and elucidated the products of its oxidation of primary amine groups of PAs when included in culture medium. These investigations formed the foundation for many polyamine-related studies and experimental procedures still performed today. This Minireview will summarize key innovative studies regarding PAs and oxidative damage, starting with those from the Tabor laboratory and including the most recent advances, with a focus on mammalian systems.

Polyamines regulate many important biological processes including gene expression, intracellular signaling, and biofilm formation. Their intracellular concentrations are tightly regulated by polyamine transport systems and biosynthetic and catabolic pathways. Spermidine/spermine N-acetyltransferases (SSATs) are catabolic enzymes that acetylate polyamines and are critical for maintaining intracellular polyamine homeostasis. These enzymes belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily and adopt a highly conserved fold found across all kingdoms of life. SpeG is an SSAT protein found in a variety of bacteria, including the human pathogen Vibrio cholerae. This protein adopts a dodecameric structure and contains an allosteric site, making it unique compared to other SSATs. Currently, we have a limited understanding of the critical structural components of this protein that are required for its allosteric behavior. Therefore, we explored the importance of two key regions of the SpeG protein on its kinetic activity. To achieve this, we created various constructs of the V. cholerae SpeG protein, including point mutations, a deletion, and chimeras with residues from the structurally distinct and non-allosteric human SSAT protein. We measured enzyme kinetic activity toward spermine for ten constructs and crystallized six of them. Ultimately, we identified specific portions of the allosteric loop and the β6-β7 structural elements that were critical for enzyme kinetic activity. These results provide a framework for further study of the structure/function relationship of SpeG enzymes from other organisms and clues toward the structural evolution of members of the GNAT family across domains of life.

Polyamine uptake transporter (PUT) plays important roles in polyamine homeostasis, but knowledge regarding PUT family genes in sweet orange (Citrus sinensis) remains elusive. Herein, our study aimed to perform a genome-wide identification of the PUT gene family in C. sinensis. A total of eight putative PUT genes (CsPUT1-CsPUT8) were identified in the sweet orange genome and distributed on three chromosomes. The CsPUT genes were divided into two major groups according to the phylogenetic tree analysis, with high similarities in protein domains and gene structure organization. The CsPUT genes were differentially expressed in different tissues, with the highest transcript levels being in the flowers and roots. Interestingly, the CsPUT genes were significantly induced by polyamines, putrescine, spermidine and spermine, indicating that CsPUT were possibly associated with intracellular polyamine transport and uptake. In addition, CsPUT showed differential expression in callus treated with ABA, cold, salt or osmotic shock. CsPUT4 was selected as a candidate for functional analysis of PUT. Overexpression of CsPUT4 elevated endogenous polyamine content and led to enhanced cold tolerance in transgenic callus cultures. Overall, these data provide valuable information for better understanding the potential biological functions of PUT genes in future.

Soil arbuscular mycorrhizal fungi (AMF) enhance the tolerance of plants against soil moisture deficit stress (SMDS), but the underlying mechanisms are still not fully understood. Polyamines (PAs) as low-molecular-weight, aliphatic polycations have strong roles in abiotic stress tolerance of plants. We aimed to investigate the effect of AMF (Funneliformis mosseae) inoculation on PAs, PA precursors, activities of PA synthases and degrading enzymes, and concentration of reactive oxygen species in the roots of trifoliate orange (Poncirus trifoliata) subjected to 15 days of SMDS. Leaf water potential and total chlorophyll levels were comparatively higher in AMF-inoculated than in non-AMF-treated plants exposed to SMDS. Mycorrhizal plants recorded a significantly higher concentration of precursors of PA synthesis such as L-ornithine, agmatine, and S-adenosyl methionine, besides higher putrescine and cadaverine and lower spermidine during the 15 days of SMDS. AMF colonization raised the PA synthase (arginine decarboxylase, ornithine decarboxylase, spermidine synthase, and spermine synthase) activities and PA-degrading enzymes (copper-containing diamine oxidase and FAD-containing polyamine oxidase) in response to SMDS. However, mycorrhizal plants showed a relatively lower degree of membrane lipid peroxidation, superoxide anion free radical, and hydrogen peroxide than non-mycorrhizal plants, whereas the difference between them increased linearly up to 15 days of SMDS. Our study concluded that AMF regulated PA homeostasis in roots of trifoliate orange to tolerate SMDS.

Robust and effective T cell-mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of de novo synthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in polyamine uptake from the environment, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular PA pool, inhibits T cell proliferation, suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine for managing inflammatory and autoimmune diseases.

Polyamine homeostasis determines T cell function., Robust and effective T cell–mediated immune responses require proper allocation of metabolic resources through metabolic pathways to sustain the energetically costly immune response. As an essential class of polycationic metabolites ubiquitously present in all living organisms, the polyamine pool is tightly regulated by biosynthesis and salvage pathway. We demonstrated that arginine is a major carbon donor and glutamine is a minor carbon donor for polyamine biosynthesis in T cells. Accordingly, the dependence of T cells can be partially relieved by replenishing the polyamine pool. In response to the blockage of biosynthesis, T cells can rapidly restore the polyamine pool through a compensatory increase in extracellular polyamine uptake, indicating a layer of metabolic plasticity. Simultaneously blocking synthesis and uptake depletes the intracellular polyamine pool, inhibits T cell proliferation, and suppresses T cell inflammation, indicating the potential therapeutic value of targeting the polyamine pool for managing inflammatory and autoimmune diseases.

Naphthalimides, such as amonafide and mitonafide in clinical trials, have been developed as antitumor agents for orthotopic tumor. However, the serious side effects in cancer patients limit their applications. Herein, a new class of polyamine-based naphthalimide conjugates 5a-5c, 7a-7b, and 11a-11b with and without the alkylation of the distant nitrogen in the polyamine chain were synthesized and the mechanism was determined. Compared with amonafide, dinitro-naphthalimide conjugate 5c with a 4,3-cyclopropyl motif preferentially accumulates in cancer cells and minimizes side effects in vitro and in vivo. More importantly, 5c at the dosage of as low as 3 mg/kg (57.97%) displays better antitumor effects than the positive control amonafide (53.27%) at 5 mg/kg in vivo. And a remarkably elevated antitumor activity and a reduced toxicity are also observed for 5c at 5 mg/kg (65.90%). The upregulated p53 and the apoptotic cells (73.50%) indicate that the mechanism of 5c to induce apoptosis may result from its enhanced DNA damage. Further investigation indicates that in addition to target DNA, 5c can modulate the polyamine homeostasis by upregulating polyamine oxidase (PAO) in a different way from that of amonafide. And also by targeting PTs overexpressed in most of cancer cells, 5c downregulates the contents of Put, Spd, and Spm, which are in favor of suppressing fast-growing tumor cells. Our study implies a promising strategy for naphthalimide conjugates to treat hepatic carcinoma with notable activities and reduced toxicities at a low dosage.

The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.

The polyamines putrescine, spermidine and spermine are essential polycations involved in the regulation of cellular proliferation. They exert dynamic effects on nucleic acids and macromolecular synthesis in vitro but their specific functions in vivo are poorly understood. Here, we have modulated the spermidine levels either by overexpressing the S-adenosylmethionine decarboxylase (samdc) gene or treating the cells with methylglyoxal-bis (guanylhydrazone) (MGBG), an inhibitor of SAMDC. In Dictyostelium, overexpression of SAMDC slowed cell proliferation, delayed development and arrested cells in the S-phase of the cell cycle. Treatment with MGBG reduced cell proliferation and stimulated development, but in samdcOE cells, it increased cell proliferation suggesting critical levels of spermidine to be important. In samdcOE cells, spermidine levels remained high throughout development but only small changes in the spermine levels were observed. Initial putrescine levels did increase but reverted to wild-type levels after the mound stage. As tight regulation of polyamine homeostasis is required, we identified genes that could be involved in its maintenance. In conclusion, we characterised samdcOE cells and observed the maintenance of polyamine homeostasis during the development of Dictyostelium cells.

Spermine, spermidine and putrescine polyamines are naturally occurring ubiquitous positively charged amines and are essential metabolites for biological functions in our life. These compounds play a crucial role in many cell processes, including cellular proliferation, growth, and differentiation. Intracellular levels of polyamines depend on their biosynthesis, transport and degradation. Polyamine levels are high in cancer cells, which leads to the promotion of tumor growth, invasion and metastasis. Targeting polyamine metabolism as an anticancer strategy is considerably rational. Due to compensatory mechanisms, a single strategy does not achieve satisfactory clinical effects when using a single agent. Combination regimens are more clinically promising for cancer chemoprevention because they work synergistically with causing little or no adverse effects due to each individual agent being used at lower doses. Moreover, bioactive substances have advantages over single chemical agents because they can affect multiple targets. In this review, we discuss anticancer strategies targeting polyamine metabolism and describe how combination treatments and effective natural active ingredients are promising therapies. The existing research suggests that polyamine metabolic enzymes are important therapeutic targets and that combination therapies can be more effective than monotherapies based on polyamine depletion.

Polyamines are small, naturally occurring polycations that are essential for normal cell growth and development. Maintaining a homeostatic level of both the polyamine pathway enzymes and the levels of the polyamines putrescine, spermidine and spermine is important because the dysregulation of the polyamine pathway has been associated with physical anomalies including cancer, Parkinson's disease, Snyder-Robinson syndrome and aging in mammals. To date, little is understood regarding how the polyamine pathway maintains polyamine homeostasis in the fly, an emerging model for polyamine studies. The aim of these studies was to better characterize how the downregulation or ablation of the polyamine biosynthetic enzymes ornithine decarboxylase, spermidine synthase and spermine synthase influence the expression of the genes coding for other polyamine pathway enzymes and the levels of the polyamines themselves utilizing the model organism, Drosophila melanogaster. We demonstrate the complexities of the polyamine metabolic pathway and show that downregulation of any of these polyamine pathway enzymes can lead to an array of changes in other parts of the pathway as well as alter the cellular levels of polyamines themselves. These data highlight the complex nature of regulation of this pathway and underscore the need to better understand its self-regulation in this and other model systems.

Free polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower (Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes – arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) – as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism.

Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis. Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na(+) concentration, and Cl(-) concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc, thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha-dl-difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock on active Cl(-) secretion. Spermidine partially reduced the decrease of short-circuit current (Isc) and membrane conductance (Gt) produced by hypotonic exposure. These data suggest polyamines blunt the hypotonic inhibition of NaCl secretion and may lead to early apoptosis of seawater ionocytes and their replacement by FW-type ionocytes.

Adverse conditions and biotic strain can lead to significant losses and impose limitations on plant yield. Polyamines (PAs) serve as regulatory molecules for both abiotic/biotic stress responses and cell protection in unfavourable environments. In this work, the transcription pattern of 24 genes orchestrating PA metabolism was investigated in Cucumber Mosaic Virus or Potato Virus Y infected and cold stressed tomato plants. Expression analysis revealed a differential/pleiotropic pattern of gene regulation in PA homeostasis upon biotic, abiotic or combined stress stimuli, thus revealing a discrete response specific to diverse stimuli: (i) biotic stress-influenced genes, (ii) abiotic stress-influenced genes, and (iii) concurrent biotic/abiotic stress-regulated genes. The results support different roles for PAs against abiotic and biotic stress. The expression of several genes, significantly induced under cold stress conditions, is mitigated by a previous viral infection, indicating a possible priming-like mechanism in tomato plants pointing to crosstalk among stress signalling. Several genes and resulting enzymes of PA catabolism were stimulated upon viral infection. Hence, we suggest that PA catabolism resulting in elevated H2O2 levels could mediate defence against viral infection. However, after chilling, the activities of enzymes implicated in PA catabolism remained relatively stable or slightly reduced. This correlates to an increase in free PA content, designating a per se protective role of these compounds against abiotic stress.

Background Genetic susceptibility of SLE in general is attributed to the overall risk of multiple common variants that each confers a small effect. However, in few cases, highly penetrant single gene variants have been reported as monogenic forms of SLE. To explore novel risk variants, we carried out whole-exome sequencing to identify underlying monogenic causes from two multiplex families that each family has two boys with childhood onset lupus nephritis. Methods We sequenced the whole exome of lupus patients and their parents using the Illumina’s instrument Hiseq2000. We conducted variant calling and annotation using the Genome Analysis Toolkit GATK and ANNOVAR, respectively. Our findings of exome-seq was confirmed using the Sanger sequencing. Results Using bioinformatics, we focused only on potential loss-of-function variants. In addition, by using the recessive inheritance model and allele frequency 2 00 000 individuals). In one family, the SAT1 frameshift mutation was transmitted from the mother to the two sons affected with SLE but not to the unaffected son. Conclusions We identified SAT1 as a novel gene associated with monogenic lupus. SAT1 encodes the spermidine/spermine-N1-acetyltransferase (SSAT), a rate-limiting enzyme that regulates the catabolism of polyamine. We hypothesize that loss-of-function SAT1 variants may cause dysregulated polyamine homeostasis which confers risk of SLE.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym‐Homospermidine is the dominant polyamine in the filamentous, N2‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst‐specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena., Work was supported by grants number BFU2014‐56757‐P and BFU2017‐88202‐P from Agencia Estatal de Investigación, Spain, co‐financed by the European Regional Development Grant (EF) and by a seed grant from UT Southwestern Medical Center (AJM).

Polyamines are organic polycations essential for cell growth and differentiation; their aberrant accumulation is often associated with diseases, including many types of cancer. To maintain polyamine homeostasis, the catalytic activity and protein abundance of ornithine decarboxylase (ODC), the committed enzyme for polyamine biosynthesis, are reciprocally controlled by the regulatory proteins antizyme isoform 1 (Az1) and antizyme inhibitor (AzIN). Az1 suppresses polyamine production by inhibiting the assembly of the functional ODC homodimer and, most uniquely, by targeting ODC for ubiquitin-independent proteolytic destruction by the 26S proteasome. In contrast, AzIN positively regulates polyamine levels by competing with ODC for Az1 binding. The structural basis of the Az1-mediated regulation of polyamine homeostasis has remained elusive. Here we report crystal structures of human Az1 complexed with either ODC or AzIN. Structural analysis revealed that Az1 sterically blocks ODC homodimerization. Moreover, Az1 binding triggers ODC degradation by inducing the exposure of a cryptic proteasome-interacting surface of ODC, which illustrates how a substrate protein may be primed upon association with Az1 for ubiquitin-independent proteasome recognition. Dynamic and functional analyses further indicated that the Az1-induced binding and degradation of ODC by proteasome can be decoupled, with the intrinsically disordered C-terminal tail fragment of ODC being required only for degradation but not binding. Finally, the AzIN-Az1 structure suggests how AzIN may effectively compete with ODC for Az1 to restore polyamine production. Taken together, our findings offer structural insights into the Az-mediated regulation of polyamine homeostasis and proteasomal degradation.

Polyamines (PAs) are ubiquitous polycations involved in many physiological processes in plants, including somatic embryogenesis, cellular growth and stress reactions. In the present study, we focus on the consequences in PA metabolism caused by polyethylene glycol (PEG) in proembryogenic Scots pine (Pinus syl- vestris L.) liquid cultures. The growth and viability of the cell masses and changes in PA concentrations and phenolic secondary metabolites were investigated under control, 5 and 10 % PEG treatments. The effect of osmotic stress responses was investigated at the gene expression level including stress, cell division, programmed cell death and PA-related genes and PA metabolites. Moreover, the expression of ethylene and proline biosynthesis genes and phenylalanine ammonia lyase and stilbene synthase (psSTS) was analyzed. Under osmotic stress conditions, we found a consistent pattern of endogenous PAs in Scots pine proembryogenic cells. However, accumulation of free spermine (Spm) and methyl putrescine under osmotic stress might indicate their specific role in stress protection. Expression of polyamine oxidase was down-regulated under osmotic stress, suggesting the role of PA catabolism in regulation of Spm levels. Scots pine proliferating proembryogenic cells are in a developmentally undiffer- entiated stage where the content of secondary metabolites is generally low. However, in the present study the total content of phenolic compounds increased but the biosyn- thesis of phenylpropanoids and stilbenes, generally con- sidered as stress-protective molecules, was not affected by osmotic stress.

The polyamines are important for a variety of cellular functions, including cell growth. Their intracellular concentrations are controlled by a complex network of regulatory mechanisms, in which antizyme (Az) has a key role. Az reduces the cellular polyamine content by down-regulating both the enzyme catalysing polyamine biosynthesis, ornithine decarboxylase (ODC), and the uptake of polyamines. The activity of Az is repressed by the binding of a protein, named Az inhibitor (AzI), which is an enzymatically inactive homologue of ODC. Two forms of AzI have been described: AzI1, which is ubiquitous, and AzI2 which is expressed in brain and testis. In the present study, we have investigated the role of AzI1 in polyamine homeostasis and cell proliferation in breast cancer cells. The results obtained showed that the cellular content of AzI increased transiently after induction of cell proliferation by diluting cells in fresh medium. Inhibition of polyamine biosynthesis induced an even larger increase in the cellular AzI content, which remained significantly elevated during the 7-day experimental period. However, this increase was not a consequence of changes in cell cycle progression, as demonstrated by flow cytometry. Instead, the increase appeared to correlate with the cellular depletion of polyamines. Moreover, induced overexpression of AzI resulted in an increased cell proliferation with a concomitant increase in ODC activity and putrescine content. During mitosis, AzI1 was localised in a pattern that resembled that of the two centrosomes, confirming earlier observations. Taken together, the results indicate that AzI fulfils an essential regulatory function in polyamine homeostasis and cell proliferation.

Polyamines (PAs) are essential biomolecules that are known to be involved in the regulation of many plant developmental and growth processes as well as their response to different environmental stimuli. Maintaining the cellular pools of PAs or their metabolic precursors and by-products is critical to accomplish their normal functions. Therefore, the titre of PAs in the cells must be under tight regulation to enable cellular PA homeostasis. Polyamine homeostasis is hence achieved by the regulation of their input into the cellular PA pool, their conversion into secondary metabolites, their transport to other issues/organs, and their catabolism or turnover. The major contributors of input to the PA pools are their in vivo biosynthesis, interconversion between different PAs, and transport from other tissues/organs; while the output or turnover of PAs is facilitated by transport, conjugation and catabolism. Polyamine metabolic pathways including the biosynthesis, catabolism/turnover and conjugation with various organic molecules have been widely studied in all kingdoms. Discoveries on the molecular transporters facilitating the intracellular and intercellular translocation of PAs have also been reported. Numerous recent studies using transgenic approaches and mutagenesis have shown that plants can tolerate quite large concentrations of PAs in the cells; even though, at times, high cellular accumulation of PAs is quite detrimental, and so is high rate of catabolism. The mechanism by which plants tolerate such large quantities of PAs is still unclear. Interestingly, enhanced PA biosynthesis via manipulation of the PA metabolic networks has been suggested to contribute directly to increased growth and improvements in plant abiotic and biotic stress responses; hence greater biomass and productivity. Genetic manipulation of the PA metabolic networks has also been shown to improve plant nitrogen assimilation capacity, which may in turn lead to enhanced carbon assimilation. These potential benefits on top of the widely accepted role of PAs in improving plants' tolerance to biotic and abiotic stressors are invaluable tools for future plant improvement strategies.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus , the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S -adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the Ke Lrp variant in KGMA7203. KGMA7110 (0.69 ± 0.27 μM) and KGMA7203 (4.90 ± 0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28 ± 0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that Ke Lrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus .

Polyamines (PAs) and hydrogen peroxide (H2O2), the product of PA oxidation by polyamine oxidase (PAO), are potential players affecting plant growth, development and responses to abiotic/biotic stresses. Genetically modified Nicotiana tabacum plants with altered PA/H2O2 homeostasis due to over/underexpression of the ZmPAO gene (S-ZmPAO/AS-ZmPAO, respectively) were assessed under heat stress (HS). Underexpression of ZmPAO correlates with increased thermotolerance of the photosynthetic machinery and improved biomass accumulation, accompanied by enhanced levels of the enzymatic and non-enzymatic antioxidants, whereas ZmPAO overexpressors exhibit significant impairment of thermotolerance. These data provide important clues on PA catabolism/H2O2/thermotolerance, which merit further exploitation.

Amine oxidase copper-containing 1 (AOC1; formerly known as amiloride-binding protein 1) is a secreted glycoprotein that catalyzes the degradation of putrescine and histamine. Polyamines and their diamine precursor putrescine are ubiquitous to all organisms and fulfill pivotal functions in cell growth and proliferation. Despite the importance of AOC1 in regulating polyamine breakdown, very little is known about the molecular mechanisms that control its expression. We report here that the Wilms tumor protein, WT1, which is necessary for normal kidney development, activates transcription of the AOC1 gene. Expression of a firefly luciferase reporter under control of the proximal AOC1 promoter was significantly enhanced by co-transfection of a WT1 expression construct. Binding of WT1 protein to a cis-regulatory element in the AOC1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Antisense inhibition of WT1 protein translation strongly reduced Aoc1 transcripts in cultured murine embryonic kidneys and gonads. Aoc1 mRNA levels correlated with WT1 protein in several cell lines. Double immunofluorescent staining revealed a co-expression of WT1 and AOC1 proteins in the developing genitourinary system of mice and rats. Strikingly, induced changes in polyamine homeostasis affected branching morphogenesis of cultured murine embryonic kidneys in a developmental stage-specific manner. These findings suggest that WT1-dependent control of polyamine breakdown, which is mediated by changes in AOC1 expression, has a role in kidney organogenesis.

Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4 h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.

Introduction Polyamines play a fundamental role during embryogenesis by regulating cell growth and proliferation and by interacting with RNA, DNA and protein. The polyamine pools are regulated by metabolism and uptake from exogenous sources. The use of certain inhibitors of polyamine synthesis causes similar defects to those seen in alcohol exposure e.g. retarded embryo growth and endothelial cell sprouting. Methods CD-1 mice received two intraperitoneal injections of 3 g/kg ethanol at 4 h intervals 8.75 days post coitum (dpc). The fetal head, trunk, yolk sac and placenta were collected at 9.5 and 12.5 dpc and polyamine concentrations were determined. Results No measurable quantity of polyamines could be detected in the embryo head at 9.5 dpc, 12 h after ethanol exposure. Putrescine was not detectable in the trunk of the embryo at that time, whereas polyamines in yolk sac and placenta were at control level. Polyamine deficiency was associated with slow cell growth, reduction in endothelial cell sprouting, an altered pattern of blood vessel network formation and consequently retarded migration of neural crest cells and growth restriction. Discussion Our results indicate that the polyamine pools in embryonic and extraembryonic tissues are developmentally regulated. Alcohol administration, at the critical stage, perturbs polyamine levels with various patterns, depending on the tissue and its developmental stage. The total absence of polyamines in the embryo head at 9.5 dpc may explain why this stage is so vulnerable to the development of neural tube defect, and growth restriction, the findings previously observed in fetal alcohol syndrome.

The translational efficiency of an mRNA can be modulated by elements located in the 5'-untranslated region. The flavin-containing polyamine oxidases catabolize oxidative deamination of spermidine and spermine, thus contributing to polyamine homeostasis as well as diverse biological processes through their reaction products. In this study, we characterized the uORF of AtPAO2 gene using the GUS reporter gene. Transgenic lines harboring the native AtPAO2 promoter or the constitutive CaMV 35S promoter show that the uORF negatively affects GUS expression. Exogenous applications of PAs positively modulate GUS expression, thus alleviating the negative effect of AtPAO2 uORF, while treatments with MGBG inhibitor show an opposite effect. Our data suggest that AtPAO2 uORF regulatory mechanism is modulated by polyamines. In addition, we present a comparative in silico study of the uORFs identified in several plant transcripts encoding polyamine oxidases in both mono- and dicotyledonous plants as well as in the Bryophyte Physcomitrella patens. The polyamine oxidase uORF-encoded peptides are conserved among families and share conserved features such as their position, length, and amino acid sequence. Our findings provide new insights into the regulatory mechanism of polyamine oxidase genes and encourage further exploration to assess the biological significance of uORFs in the polyamine catabolic pathway.

Ornithine decarboxylase antizyme 1 (OAZ1) regulates both polyamine biosynthesis and transport and acts as a tumor suppressor because of its functions in the regulation of cell proliferation, apoptosis and growth. However, the roles of OAZ1 in the regulation of polyamine metabolism, cell proliferation, apoptosis and hormone receptor expression in granulosa cells (GCs) from geese have not been determined. To define the roles of OAZ1 in primary GCs, we constructed and transfected an Oaz1 overexpression vector with a thymidine deletion into primary GCs. Oaz1 overexpression induced 65 and 30% decreases in the putrescine and spermidine contents, respectively, 225% increase in the spermine content and up-regulated the expression levels of genes associated with polyamine metabolism (but Oaz2). The Ccnd1, Pcna, Bax and Caspase 3 mRNA expression levels in GCs overexpressing Oaz1 were significantly higher than the expression levels in the vehicle group (p

Diabetic Retinopathy (DR), is a significant public health issue and the leading cause of blindness in working-aged adults worldwide. The vision loss associated with DR affects patients' quality of life and has negative social and psychological effects. In the past, diabetic retinopathy was considered as a vascular disease; however, it is now recognized to be a neuro-vascular disease of the retina. Current therapies for DR, such as laser photocoagulation and anti-VEGF therapy, treat advanced stages of the disease, particularly the vasculopathy and have adverse side effects. Unavailability of effective treatments to prevent the incidence or progression of DR is a major clinical problem. There is a great need for therapeutic interventions capable of preventing retinal damage in DR patients. A growing body of evidence shows that neurodegeneration is an early event in DR pathogenesis. Therefore, studies of the underlying mechanisms that lead to neurodegeneration are essential for identifying new therapeutic targets in the early stages of DR. Deregulation of the polyamine metabolism is implicated in various neurodegenerative diseases, cancer, renal failure, and diabetes. Spermine Oxidase (SMOX) is a highly inducible enzyme, and its dysregulation can alter polyamine homeostasis. The oxidative products of polyamine metabolism are capable of inducing cell damage and death. The current review provides insight into the SMOX-regulated molecular mechanisms of cellular damage and dysfunction, and its potential as a therapeutic target for diabetic retinopathy. Structural and functional changes in the diabetic retina and the mechanisms leading to neuronal damage (excitotoxicity, loss of neurotrophic factors, oxidative stress, mitochondrial dysfunction etc.) are also summarized in this review. Furthermore, existing therapies and new approaches to neuroprotection are discussed.

Polyamines (PAs) are ubiquitous aliphatic amines important and, in many cases, essential for plant growth, abiotic stress response, and tolerance. Here we provide evidence for genetic reprogramming of PA homeostasis that occurs during the de-etiolation, maturation, and senescence of the primary leaf in Hordeum vulgare (barley). We analyzed expression levels of key genes in the anabolic and catabolic branches of PA metabolism throughout the life cycle of etioplasts, at all steps of the functional assembly of the photosynthetic apparatus, and during leaf senescence. The changes in the total PAs titer of the leaf were followed throughout the different developmental stages. Furthermore, we align all three stages of the photosynthetic performance (rapid light-dependent de-etiolation, phase of optimal efficiency, and senescence-induced deterioration) with the changes in PA homeostasis. Finally, we focus on two phases during aging (early and late senescence) and we present their bioenergetic (for example, PSII maximal efficiency, ATPase conductivity) and genetic profiles, with emphasis on sensitive parameters that describe this process for the photosynthetic apparatus and PA metabolism, respectively. In conclusion, the fine tuning of PA homeostasis is regulated by the simultaneous genetic reprogramming of the anabolic and catabolic branches of PA metabolism and adjusts all the developmental changes from de-etiolation to maturation and senescence.

Polyamine metabolism is intimately linked to the physiological state of the cell. Low polyamines levels promote growth cessation, while increased concentrations are often associated with rapid proliferation or cancer. Delicately balanced biosynthesis, catabolism, uptake and excretion are very important for maintaining the intracellular polyamine homeostasis, and deregulated polyamine metabolism is associated with imbalanced metabolic red/ox state. Although many cellular targets of polyamines have been described, the precise molecular mechanisms in these interactions are largely unknown. Polyamines are readily interconvertible which complicate studies on the functions of the individual polyamines. Thus, non-metabolizable polyamine analogues, like carbon-methylated analogues, are needed to circumvent that problem. This review focuses on methylated putrescine, spermidine and spermine analogues in which at least one hydrogen atom attached to polyamine carbon backbone has been replaced by a methyl group. These analogues allow the regulation of both metabolic and catabolic fates of the parent molecule. Substituting the natural polyamines with methylated analogue(s) offers means to study either the functions of an individual polyamine or the effects of altered polyamine metabolism on cell physiology. In general, gem-dimethylated analogues are considered to be non-metabolizable by polyamine catabolizing enzymes spermidine/spermine-N¹-acetyltransferase and acetylpolyamine oxidase and they support short-term cellular proliferation in many experimental models. Monomethylation renders the analogues chiral, offering some advantage over gem-dimethylated analogues in the specific regulation of polyamine metabolism. Thus, methylated polyamine analogues are practical tools to meet existing biological challenges in solving the physiological functions of polyamines.

Polyamines are ubiquitous organic cations implicated in many physiological processes. Because they are positively charged at physiological pH, carrier-mediated systems are necessary for effective membrane permeation, but the identity of specific polyamine transporter proteins in eukaryotic cells remains unclear. Polyspecific organic cation transporters (OCTs) interact with many natural and xenobiotic monovalent cations and have been reported to transport dicationic compounds, including the short polyamine putrescine. In this study, we used Xenopus oocytes expressing mammalian OCT1 (SLC22A1), OCT2 (SLC22A2), or OCT3 (SLC22A3) to assess binding and transport of longer-chain polyvalent polyamines. In OCT-expressing oocytes, [(3)H]MPP(+) uptake rates were 15- to 35-fold higher than in noninjected oocytes, whereas those for [(3)H]spermidine increased more modestly above the background, up to 3-fold. This reflected up to 20-fold lower affinity for spermidine than for MPP(+); thus, K(0.5) for MPP(+) was ~50 μM in OCT1, ~170 μM in OCT2, and ~60 μM in OCT3, whereas for spermidine, K(0.5) was ~1 mM in OCT1, OCT2, and OCT3. J(max) values for MPP(+) and spermidine were within the same range, suggesting that both compounds are transported at a similar turnover rate. To gain further insight into OCT substrate specificity, we screened a selection of structural polyamine analogues for effect on [(3)H]MPP(+) uptake. In general, blocking potency increased with overall hydrophobic character, which indicates that, as for monovalent cations, hydrophobicity is a major requirement for recognition in polyvalent OCT substrates and inhibitors. Our results demonstrate that the natural polyamines are low affinity, but relatively high turnover, substrates for OCTs. The identification of OCTs as polyamine transport systems may contribute to further understanding of the mechanisms involved in polyamine homeostasis and aid in the design of polyamine-like OCT-targeted drugs.

Changes in the levels of polyamines are correlated with the activation or repression of developmental response pathways, but the role of polyamine transporters in the regulation of polyamine homeostasis and thus indirectly gene expression, has not been previously addressed. Here we show that the A. thaliana and rice transporters AtPUT5 and OsPUT1 were localized to the ER, while the AtPUT2, AtPUT3, and OsPUT3 were localized to the chloroplast by transient expression in N. benthamiana. A. thaliana plants that were transformed with OsPUT1 under the control the PUT5 promoter were delayed in flowering by 16days. In contrast, put5 mutants flowered four days earlier than WT plants. The delay of flowering was associated with significantly higher levels of spermidine and spermidine conjugates in the leaves prior to flowering. A similar delay in flowering was also noted in transgenic lines with constitutive expression of either OsPUT1 or OsPUT3. All three transgenic lines had larger rosette leaves, thicker flowering stems, and produced more siliques than wild type plants. In contrast, put5 plants had smaller leaves, thinner flowering stems, and produced fewer siliques. Constitutive expression of PUTs was also associated with an extreme delay in both plant senescence and maturation rate of siliques. These experiments provide the first genetic evidence of polyamine transport in the timing of flowering, and indicate the importance of polyamine transporters in the regulation of flowering and senescence pathways.

Plant polyamines are catabolized by two classes of amine oxidases, the copper amine oxidases (CuAOs) and the flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAOs). These enzymes differ to each other in substrate specificity, catalytic mechanism and subcellular localization. CuAOs and PAOs contribute to several physiological processes both through the control of polyamine homeostasis and as sources of biologically-active reaction products. CuAOs and PAOs have been found at high level in the cell wall of several species belonging to Fabaceae and Poaceae families, respectively, especially in tissues fated to undertake extensive wall loosening/stiffening events and/or in cells undergoing programmed cell death (PCD). Apoplastic CuAOs and PAOs have been shown to play a key role as a source of H2O2 in light- or developmentally-regulated differentiation events, thus influencing cell wall architecture and maturation as well as PCD. Moreover, growing evidence suggests a key role of intracellular CuAOs and PAOs in several facets of plant development. Here, we discuss recent advances in understanding the contribution of different CuAOs/PAOs, as well as their cross-talk with different intracellular and extracellular metabolic pathways, in tissue differentiation and organ development.

MicroRNAs (miRNAs) are non-coding regulatory molecules that play important roles in a variety of biological processes. Although a number of cold-responsive miRNAs have been computationally identified, functions and mechanisms of most miRNAs are not well understood. Herein, the function of trifoliate orange [Poncirus trifoliata (L.) Raf.] miRNA396b (ptr-miR396b) in cold tolerance and its potential regulatory module were investigated. Compared with the wild type (WT), transgenic lemon (Citrus limon) plants overexpressing ptr-MIR396b, the precursor of ptr-miR396b, displayed enhanced cold tolerance. Ptr-miR396b was experimentally confirmed to guide the cleavage of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). The overexpressing lines exhibited a reduction in ACO transcript levels and ethylene content compared with the WT, and the expression pattern of ACO was opposite to that of ptr-miR396b in response to cold stress. In addition, the transgenic lines exhibited higher levels of free polyamines and mRNA abundance of polyamine biosynthetic genes than WT plants under cold treatment, consistent with reduced reactive oxygen species (ROS) accumulation in the former. Taken together, this study demonstrates that ptr-miR396b positively regulates cold tolerance through reducing ACO transcript levels, thereby repressing ethylene synthesis and simultaneously promoting polyamine synthesis, leading to enhanced ROS scavenging. Identification of the ptr-miR396b-ACO regulatory module provides new insights into the molecular mechanism underlying the reduction of ethylene production under cold.

Experimental pancreatitis is associated with activation of polyamine catabolism. The polyamine analog bismethylspermine (Me 2 Spm) can ameliorate pancreatic injury. We investigated the roles of polyamine catabolism in remote organs during pancreatitis and explored the mechanism of polyamine catabolism by administering Me 2 Spm. Acute pancreatitis was induced by an infusion of 2 or 6% taurodeoxycholate before Me 2 Spm administration. Blood, urine and tissues were sampled at 24 and 72 h to assess multi-organ injury and polyamine catabolism. The effect of Me 2 Spm on mortality in experimental pancreatitis was tested separately. Liver putrescine levels were elevated following liver injury. Me 2 Spm increased the activity of spermidine/spermine N 1 -acetyltransferase (SSAT) and depleted the spermidine, spermine or putrescine levels. Lung putrescine levels increased, and SSAT and spermine decreased following lung injury. Me 2 Spm enhanced the activity of SSAT and decreased the spermidine and spermine levels. Renal injury was manifested as an increase in creatinine or a decrease in urine output. Decreases in kidney SSAT, spermidine or spermine and an increase in putrescine were found during pancreatitis. In the 2% taurodeoxycholate model, Me 2 Spm decreased urine output and raised plasma creatinine levels. Me 2 Spm increased SSAT and decreased polyamines. Excessive Me 2 Spm accumulated in the kidney, and greater amounts were found in the 6% taurodeoxycholate model in which this mortality was not reduced by Me 2 Spm. In the 2% taurodeoxycholate model, Me 2 Spm dose-dependently induced mortality at 72 h. Like pancreatic injury, remote organ injury in pancreatitis is associated with increased putrescine levels. However, Me 2 Spm could not ameliorate multi-organ injury. Me 2 Spm administration was associated with significant renal toxicity and induced mortality, suggesting that the current dose is too high and needs to be modified.

In plants, the meristem has to maintain a separate population of pluripotent cells that serve two main tasks, i.e., self-maintenance and organ initiation, which are separated spatially in meristem. Prior to our study, WUS and WUS-like WOX genes had been reported as essential for the development of the SAM. In this study, the consequences of gain of WOX1 function are described. Here we report the identification of an Arabidopsis gain-of-function mutant wox1-D, in which the expression level of the WOX1 (WUSCHEL HOMEOBOX 1) was elevated and subtle defects in meristem development were observed. The wox1-D mutant phenotype is dwarfed and slightly bushy, with a smaller shoot apex. The wox1-D mutant also produced small and dark green leaves, and exhibited a failure in anther dehiscence and male sterility. Molecular evidences showed that the transcription of the stem cell marker gene CLV3 was down-regulated in the meristem of wox1-D but accumulated in the other regions, i.e., in the root-hypocotyl junction and at the sites for lateral root initiation. The fact that the organ size and cell size in leaves of wox1-D are smaller than those in wild type suggests that cell expansion is possibly affected in order to have partially retarded the development of lateral organs, possibly through alteration of CLV3 expression pattern in the meristem. An S-adenosylmethionine decarboxylase (SAMDC) protein, SAMDC1, was found able to interact with WOX1 by yeast two-hybrid and pull-down assays in vitro. HPLC analysis revealed a significant reduction of polyamine content in wox1-D. Our results suggest that WOX1 plays an important role in meristem development in Arabidopsis, possibly via regulation of SAMDC activity and polyamine homeostasis, and/or by regulating CLV3 expression.

Pseudomonas putida S12 is a promising platform organism for the biological production of substituted aromatic compounds due to its extreme tolerance towards toxic chemicals. Solvent or aromatic stress tolerance may be due to membrane modifications and efflux pumps; however in general, polyamines have also been implicated in stressed cells. Previous transcriptomics results of P. putida strains producing an aromatic compound, or being exposed to the solvent toluene, indicated differentially expressed genes involved in polyamine transport and metabolism. Therefore, the metabolism of the polyamine, putrescine was investigated in P. putida S12, as no putrescine degradation pathways have been described for this strain. Via transcriptome analysis various, often redundant, putrescine-induced genes were identified as being potentially involved in putrescine catabolism via oxidative deamination and transamination. A series of knockout mutants were constructed in which up to six of these genes were sequentially deleted, and although putrescine degradation was affected in some of these mutants, complete elimination of putrescine degradation in P. putida S12 was not achieved. Evidence was found for the presence of an alternative pathway for putrescine degradation involving γ-glutamylation. The occurrence of multiple putrescine degradation routes in the solvent-tolerant P. putida S12 is indicative of the importance of controlling polyamine homeostasis, as well as of the high metabolic flexibility exhibited by this microorganism. © 2011 Elsevier B.V.

The yeast QDR3 gene encodes a plasma membrane drug : H+ antiporter of the DHA1 family that was described as conferring resistance against the drugs quinidine, cisplatin and bleomycin and the herbicide barban, similar to its close homologue QDR2. In this work, a new physiological role for Qdr3 in polyamine homeostasis is proposed. QDR3 is shown to confer resistance to the polyamines spermine and spermidine, but, unlike Qdr2, also a determinant of resistance to polyamines, Qdr3 has no apparent role in K+ homeostasis. QDR3 transcription is upregulated in yeast cells exposed to spermine or spermidine dependent on the transcription factors Gcn4, which controls amino acid homeostasis, and Yap1, the main regulator of oxidative stress response. Yap1 was found to be a major determinant of polyamine stress resistance in yeast and is accumulated in the nucleus of yeast cells exposed to spermidine-induced stress. QDR3 transcript levels were also found to increase under nitrogen or amino acid limitation; this regulation is also dependent on Gcn4. Consistent with the concept that Qdr3 plays a role in polyamine homeostasis, QDR3 expression was found to decrease the intracellular accumulation of [3H]spermidine, playing a role in the maintenance of the plasma membrane potential in spermidine-stressed cells.

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

MYC genes are deregulated in a plurality of human cancers. Through direct and indirect mechanisms, the MYC network regulates the expression of > 15% of the human genome, including both protein-coding and noncoding RNAs. This complexity has complicated efforts to define the principal pathways mediating MYC's oncogenic activity. MYC plays a central role in providing for the bioenergetic and biomass needs of proliferating cells, and polyamines are essential cell constituents supporting many of these functions. The rate-limiting enzyme in polyamine biosynthesis, ODC, is a bona fide MYC target, as are other regulatory enzymes in this pathway. A wealth of data link enhanced polyamine biosynthesis to cancer progression, and polyamine depletion may limit the malignant transformation of preneoplastic lesions. Studies with transgenic cancer models also support the finding that the effect of MYC on tumor initiation and progression can be attenuated through the repression of polyamine production. High-risk neuroblastomas (an often lethal embryonal tumor in which MYC activation is paramount) deregulate numerous polyamine enzymes to promote the expansion of intracellular polyamine pools. Selective inhibition of key enzymes in this pathway, e.g., using DFMO and/or SAM486, reduces tumorigenesis and synergizes with chemotherapy to regress tumors in preclinical models. Here, we review the potential clinical application of these and additional polyamine depletion agents to neuroblastoma and other advanced cancers in which MYC is operative. (Clin Cancer Res 2009;15(19):5956–61)

Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme-binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER-Golgi intermediate compartment (ERGIC) and in the cis-Golgi network, apparently not related to any known cell-sorting sequence. Our results rather suggest that the N-terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis.

Purpose: Polyamines are important regulators of cell growth and death. The polyamine modulated factor-1 (PMF-1) is involved in polyamine homeostasis. After identifying an enriched CpG island encompassing the PMF1 promoter, we aimed at evaluating the clinical relevance of PMF1 methylation in bladder cancer. Experimental Design: The epigenetic silencing of PMF1 by hypermethylation was tested in bladder cancer cells (n = 11) after azacytidine treatment. PMF1 methylation status was evaluated in 507 bladder tumors and 118 urinary specimens of bladder cancer patients and controls. PMF1 protein expression was analyzed by immunohistochemistry on tissue arrays containing bladder tumors for which PMF1 methylation was assessed (n = 218). Results: PMF1 hypermethylation was associated with gene expression loss, being restored in vitro by a demethylating agent. An initial set of 101 primary frozen bladder tumors served to identify PMF1 hypermethylation in 88.1% of the cases. An independent set of 406 paraffin-embedded tumors also revealed a high PMF1 methylation rate (77.6%). PMF1 methylation was significantly associated with increasing stage (P = 0.025). Immunohistochemical analyses revealed that PMF1 methylation was associated with cytoplasmic PMF1 expression loss (P = 0.032). PMF1 protein expression patterns were significantly associated with stage (P < 0.001), grade (P < 0.001), and poor overall survival using univariate (P < 0.001) and multivariate (P = 0.011) analyses. Moreover, PMF1 methylation in urinary specimens distinguished bladder cancer patients from controls (area under the curve = 0.800). Conclusion: PMF1 was identified to be epigenetically modified in bladder cancer. The association of PMF1 methylation with tumor progression and its diagnostic ability using urinary specimens support including PMF1 assessment for the clinical management of bladder cancer patients.