Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro-morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.     

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5"-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E–I and E–S complexes.     

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus<Emphasis Type="Italic"> Sclerotinia sclerotiorum</Emphasis>

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40–50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.Abbreviations AdoMetDC S-Adenosyl-methionine decarboxylase - DFMO -Difluoromethylornithine - MGBG Methylglyoxal bis-[guanyl hydrazone] - ODC Ornithine decarboxylase     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Inhibition of polyamine biosynthesis in Acanthamoeba castellanii and 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by chitosanoligosaccharide

Growth of Acanthamoeba castellaniiwas inhibited by chitosanoligosaccharide (up to 20 mg ml^–1) from the shells of crabs but was reversed by the polyamines, putrescine or spermidine, at 0.8 mM. Chitosanoligosaccharide strongly inhibited the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a key enzyme of polyamine biosynthesis, which is enhanced in tumour promotion.     

Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment

In rape leaf discs the response to osmotic stress has been found to be associated with increases in putrescine and 1,3-diaminopropane (an oxidation product of spermidine and/or spermine) and decreases in spermidine titers. In contrast, agmatine and spermine titers showed small changes while cadaverine accumulated massively. Similar results were observed in whole rape seedlings subjected to drought conditions. -DL-difluoromethylarginine (DFMA), a specific irreversible inhibitor of arginine decarboxylase, strongly inhibited polyamine accumulation in unstressed rape leaf discs, which suggested that the arginine decarboxylase pathway is constitutively involved in putrescine biosynthesis. In leaf discs treated under high osmotic stress conditions, both DFMA and DFMO (-DL-difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase) inhibited the accumulation of polyamines. Although the stressed discs treated with DFMA had a lower concentration of putrescine than those treated with DFMO, we propose that under osmotic stress the synthesis of putrescine might involve both enzymes. DFMA, but not DFMO, was also found to inhibit cadaverine formation strongly in stressed explants. The effects on polyamine biosynthesis and catabolism of cyclohexylamine, the spermidine synthase inhibitor, aminoguanidine, the diamine-oxidase inhibitor and -aminobutyric acid, a product of putrescine oxidation via diamine oxidase or spermidine oxidation via polyamine oxidase were found to depend on environmental osmotic challenges. Thus, it appears that high osmotic stress did not block spermidine biosynthesis, but induced a stimulation of spermidine oxidation. We have also demonstrated that in stressed leaf discs, exogenous ethylene, applied in the form of (2-chloroethyl) phosphonic acid or ethephon, behaves as an inhibitor of polyamine synthesis with the exception of agmatine and diaminopropane. In addition, in stressed tissues, when ethylene synthesis was inhibited by aminooxyacetic acid or aminoethoxyvinylglycine, S-adenosylmethionine utilization in polyamine synthesis was not promoted. The relationships between polyamine and ethylene biosynthesis in unstressed and stressed tissues are discussed.     

Polyamine Localization and Biosynthesis in Chemically Fractionated Rat Retina

For elucidation of polyamine localization and biosynthesis in various cell types of rat retina, the putrescine, spermidine, and spermine contents as well as the ornithine decarboxylase and S-adenosylmethionine decarboxylase activities have been measured in retinal cell layers obtained by the selective cytotoxic action of iodoacetate on photoreceptor cells and of monosodium glutamate on higher-order retinal neurons. A notable depletion only in spermine content was associated with loss of the visual cell layer. Total ornithine decarboxylase and S-adenosylmethionine decarboxylase activities per retina were significantly lower in all chemically fractionated tissue, but loss of the photoreceptor layer produced the greatest decrease. The specific activities of these enzymes did not show marked changes in rat retinas deprived of inner neurons. The data support the suggestions that polyamine synthesis, storage, and catabolism have different distributions in the retinal layers and that the spermine levels and the high value of the spermine/spermidine molar ratio might depend essentially on the proportion of rods to cones.     

Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.     

Short-term polyamine response in TMV-inoculated hypersensitive and susceptible tobacco plants

The short-term polyamine response to inoculation, with tobacco mosaic virus (TMV), of TMV-inoculated NN (hypersensitive) and nn (susceptible) plants of Nicotiana tabacum (L.) cv. Samsun was investigated. Free and conjugated polyamine concentrations, putrescine biosynthesis, evaluated through arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) activities, and putrescine oxidation, via diamine oxidase (DAO) activity, were analysed during the first 24 h from inoculation. Results were compared with those of mock-inoculated control plants. In NN TMV-inoculated plants undergoing the hypersensitive response (HR), free putrescine and spermidine concentrations had increased after 5 h compared with controls; polyamine conjugates also tended to increase compared with controls. In both virus- and mock-inoculated plants, ADC and ODC activities generally increased whereas DAO activity, which was present in controls, was detectable only in traces in inoculated tissues.
In TMV-infected susceptible plants, free putrescine and spermidine concentrations were lower at 5 h relative to controls, as were polyamine conjugates. No differences were revealed in ADC and ODC activities whereas DAO activity was not detectable. These results further support the hypothesis that polyamines are involved in the response of tobacco to TMV and that, only a few hours after inoculation, the response of hypersensitive plants is distinct from that of susceptible ones.     

Polyamine metabolism and cancer

Polyamines are aliphatic cations present in all cells. In normal cells, polyamine levels are intricately controlled by biosynthetic and catabolic enzymes. The biosynthetic enzymes are ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. The catabolic enzymes include spermidine/spermine acetyltransferase, flavin containing polyamine oxidase, copper containing diamine oxidase, and possibly other amine oxidases. Multiple abnormalities in the control of polyamine metabolism and uptake might be responsible for increased levels of polyamines in cancer cells as compared to that of normal cells. This review is designed to look at the current research in polyamine biosynthesis, catabolism, and transport pathways, enumerate the functions of polyamines, and assess the potential for using polyamine metabolism or function as targets for cancer therapy.     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.