Escherichia coli cytotoxic necrotizing factors (CNFs) and Bordetella dermonecrotic toxin (DNT) have been recently found to comprise a novel family of dermonecrosis-inducing toxins which activate the small GTPases of the Rho family. They are single chain polypeptides consisting of an N-terminal domain responsible for binding to target cells and a C-terminal catalytic domain. CNFs (CNF1 and 2) and DNT share in the catalytic domain about 30% identical residues and a consensus sequence where the catalytically active center Cys resides. Both toxins deamidate Rho and other members of the Rho family, Rac and Cdc42, at Gln in the switch II region, which plays an important role in their GTPase activity. DNT, in addition, catalyzes a cross-link of the Gln of the GTPases with ubiquitous polyamines such as putrescine, spermidine, and spermine. The deamidation and the polyamination result in abrogation of the GTPase activity, and in addition, the polyamination endows Rho with the ability to interact with a downstream effector, ROCK, in a GTP-independent manner. These effects render the GTPases constitutively active, which underlies the toxicities of CNFs and DNT.

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.

Putrescine and spermidine uptake in carrot (Daucus carota L., cv "Tip top") protoplasts and isolated vacuoles was studied. Protoplasts and vacuoles accumulated polyamines very quickly, with maximum absorption within 1 to 2 minutes. The insertion of a washing layer containing 100 millimolar unlabeled putrescine or spermidine did not change this pattern, but strongly reduced the uptake of putrescine and spermidine in protoplasts and in vacuoles. The dependence of spermidine uptake on the external concentration was linear up to the highest concentrations tested in protoplasts, while that in vacuoles showed saturation kinetics below 1 millimolar (K(m) = 61.8 micromolar) and a linear component from 1 to 50 millimolar. Spermidine uptake in protoplasts increased linearly between pH 5.5 and 7.0, while there was a distinct optimum at pH 7.0 for vacuoles. Preincubation of protoplasts with 1 millimolar Ca(2+) affected only surface binding but not transport into the cells. Nonpermeant polycations such as La(3+) and polylysine inhibited spermidine uptake into protoplasts. Compartmentation studies showed that putrescine and spermidine were partly vacuolar in location and that exogenously applied spermidine could be recovered inside the cells. The characteristics of the protoplast and vacuolar uptake system induce us to put forward the hypothesis of a passive influx of polyamines through the plasmalemma and of the presence of a carrier-mediated transport system localized in the tonoplast.

The polyamine biosynthetic pathway is an important drug target for the treatment of human African trypanosomiasis (HAT), raising interest in understanding polyamine function and their mechanism of regulation. Polyamine levels are tightly controlled in mammalian cells, but similar regulatory mechanisms appear absent in trypanosomes. Instead trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes a key step in the biosynthesis of the polyamine spermidine, is activated by dimerization with an inducible protein termed prozyme. Prozyme is an inactive paralog of the active AdoMetDC enzyme that evolved by gene duplication and is found only in the trypanosomatids. In Trypanosoma brucei, AdoMetDC activity appears to be controlled by regulation of prozyme protein levels, potentially at the translational level.

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37 degrees C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, or alanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi) from calf brain reconstituted into phospholipid vesicles. The order of potency was spermine greater than spermidine greater than putrescine = cadaverine greater than tyramine. The physiological relevance of this observation was assessed, showing the same order of potency of polyamines in the stimulation of peritoneal and tracheal rat mast cells. The activation of rat mast cells by polyamines was inhibited by benzalkonium chloride or by a 2 h pretreatment of the cells with pertussis toxin. The increase in inositol phosphates evoked by polyamines was also inhibited by pertussis toxin. Therefore we propose that intracellular polyamines might control the basal level of second messengers and modulate extracellular signals transduced through G-protein-coupled receptors.

Gram-negative polychlorophenol-degrading bacterial strains KF1T (T = type strain), KF3, and NKF1, which were described previously as Pseudomonas saccharophila strains, were studied by chemotaxonomic, genetic, and physiological methods and by electron microscopy and compared with selected xenobiotic compound-degrading bacteria. These strains contained sphingolipids with d-18:0, d-20:1, and d-21:1 as the main dihydrosphingosines, ubiquinone 10 as the main respiratory quinone, and spermidine as the major polyamine, and the DNA G + C content was 66 mol%. The cellular fatty acids included about 60% octadecenoic acid, 9% 2-hydroxymyristic acid, 14% cis-9-hexadecenoic acid, and 10% hexadecanoic acid. These strains exhibited less than 97% 16S ribosomal DNA sequence similarity to all of the other taxa studied. In the DNA-DNA reassociation studies the highest levels of reassociation between these strains and previously described species were less than 40%. Thin sections of cells of strains KF1T, KF3, and NKF1 were examined by electron microscopy, and the results showed that the cells had peculiar concentrically arranged layered membranous blebs that extruded from the outer membrane, especially at the cell division points. On the basis of the results of this study, polychlorophenol-degrading strains KF1T, KF3, and NKF1 are considered members of a new species of the genus Sphingomonas, Sphingomonas subarctica. The polycyclic aromatic hydrocarbon-degrading organism Sphingomonas paucimobilis EPA 505 was closely related to Sphingomonas chlorophenolica as determined by chemotaxonomic, phylogenetic, and physiological criteria. The xenobiotic compound degraders Alcaligenes sp. strain A175 and Pseudomonas sp. strain BN6 were identified as members of species of the genus Sphingomonas.

Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the alpha-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N(1)-aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the alpha-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some alpha-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.

We have earlier shown that alpha-methylated spermidine and spermine analogues rescue cells from polyamine depletion-induced growth inhibition and maintain pancreatic integrity under severe polyamine deprivation. However, because alpha-methylspermidine can serve as a precursor of hypusine, an integral part of functional eukaryotic translation initiation factor 5A required for cell proliferation, and because alpha, omega-bismethylspermine can be converted to methylspermidine, it is not entirely clear whether the restoration of cell growth is actually attributable to hypusine formed from these polyamine analogues. Here, we have used optically active isomers of methylated spermidine and spermine and show that polyamine depletion-induced acute cytostasis in cultured cells could be reversed by all the isomers of the methylpolyamines irrespective of whether they served or not as precursors of hypusine. In transgenic rats with activated polyamine catabolism, all the isomers similarly restored liver regeneration and reduced plasma alpha-amylase activity associated with induced pancreatitis. Under the above experimental conditions, the (S, S)- but not the (R, R)-isomer of bismethylspermine was converted to methylspermidine apparently through the action of spermine oxidase strongly preferring the (S, S)-isomer. Of the analogues, however, only (S)-methylspermidine sustained cell growth during prolonged (more than 1 week) inhibition of polyamine biosynthesis. It was also the only isomer efficiently converted to hypusine, indicating that deoxyhypusine synthase likewise possesses hidden stereospecificity. Taken together, the results show that growth inhibition in response to polyamine depletion involves two phases, an acute and a late hypusine-dependent phase.

A systematic investigation of the impact of spermidine analogues both in vitro and in vivo is described. The study characterizes the effects of these analogues on L1210 cell growth, polyamine pools, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermidine/spermine N1-acetyltransferase, the maintenance of cellular charge, i.e., cationic equivalence associated with the polyamines and their analogues, and compares their ability to compete with spermidine for transport. The findings clearly demonstrate that the activity of the linear polyamine analogues is highly dependent on the length of the triamines and the size of the N(alpha),N(omega)-substituents. It appears that there is an optimum chain length for various activities and that the larger the N(alpha),N(omega)-alkyls, the less active the compound. Metabolic transformation including N-dealkylation of these compounds is also evaluated. While there is no monotonic relationship between chain length and the ability of the analogue to be metabolized, the dipropyl triamines are clearly more actively catabolized than the corresponding methyl and ethyl systems. A comparison of the triamines with the corresponding tetraamines is made throughout the text regarding both in vitro activity against L1210 cells and in vivo toxicity measurements, suggesting that several triamine analogues may offer therapeutic advantages over the corresponding tetraamines.

The nonsteroidal antiinflammatory drug sulindac displays chemopreventive activity in patients with familial adenomatous polyposis (FAP). Sulindac metabolites induce apoptosis in colon tumor cells, in part, by a polyamine-dependent mechanism that can be suppressed with exogenous putrescine. To determine the relevance of this mechanism in animals, we treated Apc(Min/+) mice, a model of human FAP, with sulindac alone or in combination with dietary putrescine. Sulindac increased steady-state RNA levels and enzymatic activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase and intestinal levels of monoacetylspermidine, spermidine, and spermine in the small intestine of mice. Sulindac also decreased the activity of the biosynthetic enzyme ornithine decarboxylase but not adenosylmethionine decarboxylase (AMD). Dietary putrescine increased intestinal putrescine contents, whereas the combination of dietary putrescine and sulindac yielded the highest levels of intestinal putrescine and correlated with a statistically significant reduction in AMD enzyme activity. Dietary putrescine did not statistically significantly increase tumorigenesis, although it significantly increased the grade of adenoma dysplasia (P < 0.05). The effectiveness of sulindac to suppress intestinal carcinogenesis was partially abrogated by dietary putrescine. These data suggest that sulindac exerts at least some of its anticarcinogenic effects in mice via a polyamine-dependent mechanism. Because high concentrations of putrescine can be found in certain dietary components, it may be advantageous to restrict dietary putrescine consumption in patients undergoing treatment with sulindac.

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.

Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine(4+) and spermine(3+), can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine(4+)>> spermidine(3+)>>>> cadavarine(2+) approximately putrescine(2+) approximately agmatine(2+) approximately N1-acetylspermidine(2+), indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14 omega, which encodes the Arabidopsis 14-3-3 isoform omega, indicated that loop 8 (residues 208-219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg(2+)-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg(2+) dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.

We have developed a one-step method to synthesize carbon quantum dots (CQDPAs) from biogenic polyamines (PAs) as an antibacterial agent for topical treatment of bacterial keratitis (BK). CQDs synthesized by direct pyrolysis of spermidine (Spd) powder through a simple dry heating treatment exhibit a solubility and yield much higher than those from putrescine and spermine. We demonstrate that CQDs obtained from Spds (CQDSpds) possess effective antibacterial activities against non-multidrug-resistant Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica serovar Enteritidis bacteria and also against the multidrug-resistant bacteria, methicillin-resistant S. aureus. The minimal inhibitory concentration (MIC) of CQDSpds is ∼2500-fold lower than that of spermidine alone, demonstrating their strong antibacterial capabilities. Investigation of the possible mechanisms behind the antibacterial activities of the as-synthesized CQDSpds indicates that the super-cationic CQDSpds with small size (diameter ca. 6 nm) and highly positive charge (ζ-potential ca. +45 mV) cause severe disruption of the bacterial membrane. In vitro cytotoxicity, hemolysis, hemagglutination, genotoxicity, and oxidative stress and in vivo morphologic and physiologic cornea change evaluations show the good biocompatibility of CQDSpds. Furthermore, topical ocular administration of CQDSpds can induce the opening of the tight junction of corneal epithelial cells, thereby leading to great antibacterial treatment of S. aureus-induced BK in rabbits. Our results suggest that CQDSpds are a promising antibacterial candidate for clinical applications in treating eye-related bacterial infections and even persistent bacteria-induced infections.

The natural polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM) are ubiquitous constituents of eukaryotic cells. The increase of PA in malignant and proliferating cells attracted the interest of scientists during last decades, addressing PA depletion as a new strategy to inhibit cell growth. Selective enzyme inhibitors were developed for decreasing PA metabolism and to act as chemotherapeutic anticancer agents. Indeed, the complexity of the PA homoeostasis overcomes the PA perturbation by a single enzyme to take effect therapeutically. Recently, an increasing interest has been posed on spermine-oxidase (SMO), the only catabolic enzyme able to specifically oxidise SPM. Interestingly, the absence of SPM is compatible with life, but its accumulation and degradation is lethal. Augmented SMO activity provokes an oxidative stress rendering cells prone to die, and appears to be important in the cell differentiation pathway. Extra-cellular SPM is cytotoxic, but its analogues are capable of inhibiting cell growth at low concentrations, most likely by intracellular SPM depletion. These pivotal roles seem to evoke the biological processes of stress response, wherein balance is mandatory to live or to die. Thus, altering SPM metabolism could allow a multi-tasking therapeutic strategy, addressed not only to inhibit PA metabolism. Several tetramines are presently in early phases (I and II) of clinical trials, and it will be a matter of a few more years to understand whether SPM-related therapeutic approaches would be of benefit for composite treatment protocols of cancer.

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about changes in polyamine synthesis associated with development of the ovine conceptus (embryo/fetus and associated placental membranes). We hypothesized that rates of placental polyamine synthesis were maximal during the rapid placental growth that occurs in the first half of pregnancy. This hypothesis was tested using ewes between Days 30 and 140 of gestation. Columbia cross-bred ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation (Day 0 = mating; n = 4 ewes/day) to obtain placentomes, intercotyledonary placenta, intercaruncular endometrium, and allantoic as well as amniotic fluids. The tissues were analyzed for ornithine decarboxylase (ODC) and arginase activities; arginine, ornithine, and polyamine concentrations; and polyamine synthesis using radiochemical and chromatographic methods. Maximal ODC and arginase activities and the highest rates of polyamine synthesis were observed in all tissues on Day 40 of gestation. Concentrations of ornithine and polyamines in placentomes and intercaruncular endometrium also peaked on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Amniotic fluid spermine increased progressively with advancing gestation. Results of the present study indicate metabolic coordination among the several integrated pathways that support high rates of polyamine synthesis in the placenta and endometrium during early pregnancy. Our findings may have important implications for both intrauterine growth retardation and fetal origins of diseases in adults.

Seven new aminosterols related to squalamine (8) were isolated from the liver of the dogfish shark Squalus acanthias. Their structures (1-7) were determined using spectroscopic methods, including 2D NMR and HRFABMS. These aminosterols possess a relatively invariant cholestane skeleton with a trans AB ring junction, a spermidine or spermine attached equatorially at C3, and a steroidal side-chain that may be sulfated. The structure of the lone spermine conjugate, 7 (MSI-1436), was confirmed by its synthesis from (5alpha,7alpha, 24R)-7-hydroxy-3-ketocholestan-24-yl sulfate. Some members of this family of aminosterols exhibit a broad spectrum of antimicrobial activity comparable to squalamine.

Polyamine levels in rodent tissues are regulated by the activities of three enzymes: ornithine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine/spermine N1-acetyltransferase. These enzymes are present in the cell in very small amounts, have very short half-lives, and are highly inducible. Ornithine decarboxylase was purified to homogeneity (about 10,000-fold) from androgen-treated mouse kidneys, which have enzyme levels several hundred times higher than those in other fully induced mammalian tissues. This decarboxylase could be specifically labeled either in vitro or in vivo by reaction with radioactive alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. Such covalent binding of alpha-difluoromethylornithine was used to titrate the number of molecules of the enzyme and to estimate its purity. It was also used for autoradiographic localization of the enzyme within tissues and to follow the degradation of the protein in vivo. S-Adenosylmethionine decarboxylase has been purified from rat liver and psoas muscle, and significant differences between the enzyme forms present in these tissues were observed. The rate-limiting enzyme in the interconversion of the polyamines, spermidine/spermine N1-acetyltransferase was purified more than 100,000-fold from carbon tetrachloride-induced rat liver. This acetylase acts on both spermine and spermidine to form N1-acetyl derivatives, which are then oxidized by polyamine oxidase forming spermidine and putrescine, respectively.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.

Two recently developed fluorescence cytochemical methods, specific for spermidine and spermine, were used to localize polyamines in the endocrine pancreas. The polyamines were restricted to the insulin-producing beta-cells and were mainly associated with the secretory granules. Chemical polyamine determinations carried out on isolated rat and mouse pancreatic islets revealed large amounts of polyamines. Compared with extracts of whole pancreas, the islets contained very high concentrations of spermine relative to spermidine. Biosynthesis of polyamines from [3H]ornithine or from [3H]putrescine in isolated islets was significantly stimulated at high glucose concentrations. Moreover, significant incorporation of label from [3H]putrescine was also detected in gamma-aminobutyric acid. This incorporation, however, was not stimulated by high glucose. Possible roles for polyamines associated with the secretory granules in insulin-producing cells are discussed.

The polyamines spermidine and spermine, and their precursor putrescine, are required for cell growth and cellular functions. The high levels of tissue polyamines are implicated in carcinogenesis. The major sources of exogenous polyamines are diet and intestinal luminal bacteria in gastrointestinal (GI) tissues. Both endocytic and solute carrier-dependent mechanisms have been described for polyamine uptake. Knocking down of caveolin-1 protein increased polyamine uptake in colon cancer-derived HCT116 cells. Dietary supplied putrescine was accumulated in GI tissues and liver in caveolin-1 knockout mice more than wild-type mice. Knocking out of nitric oxide synthase (NOS2), which has been implicated in the release of exogenous polyamines from internalized vesicles, abolished the accumulation of dietary putrescine in GI tissues. Under conditions of reduced endogenous tissue putrescine contents, caused by treatment with the polyamine synthesis inhibitor difluoromethylornithine (DFMO), small intestinal and colonic mucosal polyamine contents increased with dietary putrescine levels, even in mice lacking NOS2. Knocking down the solute carrier transporter SLC3A2 in HCT116-derived Hkh2 cells reduced the accumulation of exogenous putrescine and total polyamine contents in DFMO treated cells, relative to non-DFMO-treated cells. These data demonstrate that exogenous putrescine is transported into GI tissues by caveolin-1- and NOS2-dependent mechanisms, but that the solute carrier transporter SLC3A2 can function bidirectionally to import putrescine under conditions of low tissue polyamines.