Polyamines are classically known by their names of putrescine, spermine, and spermidine. They are synthesized endogenously from ornithine and are interconvertible. In addition, an exogenous supply of polyamines is provided by dietary intake and by intestinal absorption from the products of bacterial metabolism. Polyamine uptake occurs almost entirely in the gut, and afterward the various forms are metabolized in different tissues under the strict regulation of ornithine decarboxylase, which is the first enzyme involved in their synthesis. Polyamines are eliminated from the organism by means of oxidation reactions, appearing in urine in all their metabolic forms. Polyamines play an important role in regulating cell growth and proliferation, the stabilization of negative charges of DNA, RNA transcription, protein synthesis, apoptosis, and the regulation of the immune response. They are components of breast milk and might be important in neonatal gut maturation, for which reason the possible supplementation of infant formulas with these compounds is under study.

The putative translation factor eIF5A is essential for cell viability and is highly conserved from archaebacteria to mammals. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. This review focuses on the functional characterization of eIF5A. Although this protein was originally identified as a translation initiation factor, subsequent studies did not support a role for eIF5A in general translation initiation. eIF5A has also been implicated in nuclear export of HIV-1 Rev and mRNA decay, but these findings are controversial in the literature and may reflect secondary effects of eIF-5A function. Next, the involvement of eIF5A and hypusination in the control of the cell cycle and proliferation in various organisms is reviewed. Finally, recent evidence in favor of reconsidering the role of eIF5A as a translation factor is discussed. Future studies may reveal the specific mechanism by which eIF5A affects protein synthesis.

The trypanosomatid metabolite N1,N8-bis-(glutathionyl)spermidine (trypanothione) has been demonstrated to form a stable adduct with the aromatic arsenical drug melarsen oxide [p-(4,6-diamino-s-triazinyl-2-yl)aminophenyl arsenoxide]. The stability constant of the melarsen-trypanothione adduct (Mel T) has been determined to be 1.05 x 10(7) M-1. When bloodstream Trypanosoma brucei are incubated with either melarsen oxide or the 2,3-dimercaptopropanol adduct of melarsen oxide (melarsoprol), Mel T is the only arsenical derivative detectable in acid-soluble extracts of the cells. Trypanothione may therefore be regarded as a primary target for aromatic arsenical derivatives against African trypanosomes. The selective toxic action of these compounds might arise through sequestration of intracellular trypanothione in the form of Mel T, or Mel T itself may be toxic within the cell. The latter possibility is illustrated by the finding that Mel T is an inhibitor of trypanothione reductase from T. brucei (Ki = 9.0 microM)--an enzyme that is central to the regulation of the thiol/disulfide redox balance in the parasite and absent from the host.

Aminopropyltransferases use decarboxylated S-adenosylmethionine as an aminopropyl donor and an amine acceptor to form polyamines. This review covers their structure, mechanism of action, inhibition, regulation and function. The best known aminopropyltransferases are spermidine synthase and spermine synthase but other members of this family including an N(1)-aminopropylagmatine synthase have been characterized. Spermidine synthase is an essential gene in eukaryotes and is very widely distributed. Key regions in the active site, which are very highly conserved, were identified by structural studies with spermidine synthase from Thermotoga maritima bound to S-adenosyl-1,8-diamino-3-thiooctane, a multisubstrate analog inhibitor. A general mechanism for catalysis by aminopropyltransferases can be proposed based on these studies. Spermine synthase is less widely distributed and is not essential for growth in yeast. However, Gy mice lacking spermine synthase have multiple symptoms including a profound growth retardation, sterility, deafness, neurological abnormalities and a propensity to sudden death, which can all be prevented by transgenic expression of spermine synthase. A large reduction in spermine synthase in human males due to a splice site variant causes Snyder-Robinson syndrome with mental retardation, hypotonia and skeletal abnormalities.

Escherichia coli single-strand binding protein (SSB) or phage T4 gene 32 protein reduced the amount of recA protein required to catalyze the formation of D loops from double-stranded DNA and homologous single-stranded fragments. Neither SSB nor gene 32 protein alone catalyzed the formation of D loops, and excessive amounts of either protein, amounts that were sufficient to saturate the single strands, inhibited the formation of D loops completely. Both the stimulatory activity and the inhibitory activity of SSB resisted boiling, which is consistent with the known thermal stability of SSB, whereas the gene 32 protein was inactivated by heating. The formation of D loops in the presence of both recA protein and SSB required homologous DNA and ATP. Spermidine aided the combined action of SSB and recA protein in forming D loops, but Mg2+ alone was sufficient as a counterion.

Sites on an fd DNA template which terminate synthesis catalyzed by each of four forms of Escherichia coli DNA polymerase III have been identified at single nucleotide resolution. Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3'-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis. Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination. The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing. Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local hydrogen-bonding interactions. The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme. The occurrence of termination sites is markedly affected by the inclusion of spermidine or Escherichia coli single-stranded DNA binding protein in the reaction mixtures. Additionally, a nucleotide composition specificity of pause sites has been observed.

Polyamines, among other functions, are considered to act as a free radical scavenger and antioxidant. The quinolinic acid (QA), sodium nitroprusside (SNP) and iron (Fe+2) stimulate production of free radicals and lipid peroxidation. In the present study, we investigated the free radical and/or aldehyde scavenger effects of polyamines spermine and spermidine on thiobarbituric acid reactive species (TBARS) production induced by QA, SNP, Fe+2/EDTA system and free Fe2+ in rat brain. Spermine and spermidine inhibited QA-induced TBARS production; however spermine was a better antioxidant than spermidine. Spermine also inhibited SNP-, Fe+2/EDTA- and free Fe2+-induced TBARS production, but had a modest effect. Spermidine, in turn, also discretely inhibited SNP-, Fe+2/EDTA- and free Fe2+-induced TBARS production. In the presence of MK-801, QA-induced TBARS production was considerably more inhibited by polyamines. In addition, arcaine does not affect the reducer effect of polyamines. The present findings suggest that the observed effects of polyamines are not related to the activation of NMDA receptor but with their antioxidant and free radical scavenger properties.

DNA microarray analysis was used to analyze the transcriptional profile of HCT116 colorectal cancer cells that were treated with 5-fluorouracil (5-FU) or oxaliplatin and selected for resistance to these agents. Bioinformatic analyses identified sets of genes that were constitutively dysregulated in drug-resistant cells and transiently altered following acute exposure of parental cells to drug. We propose that these genes may represent molecular signatures of sensitivity to 5-FU and oxaliplatin. Using real-time reverse transcription-PCR (RT-PCR), the robustness of our microarray data was shown with a strong overall concordance of expression trends for>> or =82% (oxaliplatin) and>> or =85% (5-FU) of a representative subset of genes. Furthermore, strong correlations between the microarray and real-time RT-PCR measurements of average fold changes in gene expression were observed for both the 5-FU (R(2)>> or = 0.73) and oxaliplatin gene sets (R(2)>> or = 0.63). Functional analysis of three genes identified in the microarray study [prostate-derived factor (PDF), calretinin, and spermidine/spermine N(1)-acetyl transferase (SSAT)] revealed their importance as novel regulators of cytotoxic drug response. These data show the power of this novel microarray-based approach to identify genes which may be important markers of response to treatment and/or targets for therapeutic intervention.

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.

Pseudomonas aeruginosa is known to break down arginine by the arginine deiminase pathway. An additional pathway has now been found whereby arginine is converted to putrescine with agmatine and N-carbamoylputrescine as intermediates. The following enzyme activities belonging to this pathway were detected in crude extracts: arginine decarboxylase (EC 4.1.1.19), which catalyses the release of CO2 from arginine to give agmatine; agmatine deiminase (EC 3.5.3.12), which degrades agmatine to N-carbamoylputrescine; and N-carbamoylputrescine amidinohydrolase (EC 3.5.3.-), which then removes the ureido group of carbamoylputrescine. In crude extracts, arginine decarboxylase activity was stimulated by pyridoxal phosphate, Mg2+ and by the products of the catabolic pathway, putrescine and spermidine. Growth of P. aeruginosa on arginine as the sole carbon and nitrogen source markedly increased the activity of arginine decarboxylase. Agmatine and N-carbamoylputrescine induced the synthesis of agmatine deiminase and N-carbamoylputrescine hydrolase. Addition of succinate or citrate to medium containing arginine or agmatine led to repression of the enzymes involved in the arginine decarboxylase pathway. Moreover, the repression of agmatine deiminase and N-carbamoylputrescine hydrolase was further increased when P. aeruginosa was grown in media with agmatine plus glutamine or agmatine plus succinate and ammonia. This suggests that the expression of the agmatine pathway may be regulated by carbon catabolite repression as well as nitrogen catabolite repression.

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A(1), an inhibitor of the vacuolar H(+)-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

D-myo-Inositol 1,4,5-trisphosphate has been previously demonstrated to act as a second messenger for the hormonal mobilization of intracellular calcium in rat liver. In this study, the breakdown of D-myo-inositol 1,4,5-trisphosphate by a phosphatase activity was characterized. Using partially purified subcellular fractions, it was found that D-myo-inositol 1,4,5-trisphosphate phosphatase (I-P3ase) specific activity was highest in the plasma membrane fraction, while D-myo-inositol 1,4-bisphosphate phosphatase specific activity was highest in the cytosolic and microsomal fractions. The plasma membrane I-P3ase was Mg2+-dependent with optimal activity observed at 0.5-1.5 mM free Mg2+. The enzyme had a neutral pH optimum, suggesting that it was neither an acid nor alkaline phosphatase. Neither LiCl nor NaF inhibited the I-P3ase activity. However, both L-cysteine and dithiothreitol stimulated the activity 2-fold. Spermine (2.0 mM) inhibited the I-P3ase activity by 50%, while putrescine and spermidine had little or no effect.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.

Results of recent biochemical and electrophysiological studies have suggested that a recognition site for polyamines exists as part of the NMDA receptor complex. This site appears to be distinct from previously described binding sites for glutamate, glycine, Mg++,Zn++, and open-channel blockers such as MK-801. The endogenous polyamines spermine and spermidine increase the binding of open-channel blockers and increase NMDA-elicited currents in cultured neurons. These polyamines have been termed agonists at the polyamine recognition site. Studies of the effects of natural and synthetic polyamines on the binding of [3H]MK-801 and on NMDA-elicited currents in cultured neurons have led to the identification of compounds classified as partial agonists, antagonists, and inverse agonists at the polyamine recognition site. Polyamines have also been found to affect the binding of ligands to the recognition sites for glutamate and glycine. However, these effects may be mediated at a site distinct from that at which polyamines act to modulate the binding of open-channel blockers. Endogenous polyamines may modulate excitatory synaptic transmission by acting at the polyamine recognition site of the NMDA receptor. This site could represent a novel therapeutic target for the treatment of ischemia-induced neurotoxicity, epilepsy, and neurodegenerative diseases.

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

Polyamines are small ubiquitous molecules that have been involved in nearly all developmental processes, including the stress response. Nevertheless, no direct evidence of a role of polyamines in the wound response has been described. We have studied the expression of genes involved in polyamine biosynthesis in response to mechanical injury. An increase in the expression of the arginine decarboxylase 2 (ADC2) gene in response to mechanical wounding and methyl jasmonate (JA) treatment in Arabidopsis was detected by using DNA microarray and RNA gel-blot analysis. No induction was observed for the ADC1 gene or other genes coding for spermidine and spermine synthases, suggesting that ADC2 is the only gene of polyamine biosynthesis involved in the wounding response mediated by JA. A transient increase in the level of free putrescine followed the increase in the mRNA level for ADC2. A decrease in the level of free spermine, coincident with the increase in putrescine after wounding, was also observed. Abscisic acid effected a strong induction on ADC2 expression and had no effect on ADC1 expression. Wound-induction of ADC2 mRNA was not prevented in the JA-insensitive coi1 mutant. The different pattern of expression of ADC2 gene in wild-type and coi1 mutant might be due to the dual regulation of ADC2 by abscisic acid and JA signaling pathways. This is the first direct evidence of a function of polyamines in the wound-response, and it opens a new aspect of polyamines in plant biology.

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone.

OBJECTIVE

Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.

Anxiety and stress-related disorders, namely posttraumatic stress disorder (PTSD), generalized anxiety disorder (GAD), obsessive-compulsive disorder (ODC), social and specific phobias, and panic disorder, are a major public health issue. A growing body of evidence suggests that glutamatergic neurotransmission may be involved in the biological mechanisms underlying stress response and anxiety-related disorders. The glutamatergic system mediates the acquisition and extinction of fear-conditioning. Thus, new drugs targeting glutamatergic neurotransmission may be promising candidates for new pharmacological treatments. In particular, N-methyl-d-aspartate receptors (NMDAR) antagonists (AP5, AP7, CGP37849, CGP39551, LY235959, NPC17742, and MK-801), NMDAR partial agonists (DCS, ACPC), α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) antagonists (topiramate), and several allosteric modulators targeting metabotropic glutamate receptors (mGluRs) mGluR1, mGluR2/3, and mGluR5, have shown anxiolytic-like effects in several animal and human studies. Several studies have suggested that polyamines (agmatine, putrescine, spermidine, and spermine) may be involved in the neurobiological mechanisms underlying stress-response and anxiety-related disorders. This could mainly be attributed to their ability to modulate ionotropic glutamate receptors, especially NR2B subunits. The aim of this review is to establish that glutamate neurotransmission and polyaminergic system play a fundamental role in the onset of anxiety-related disorders. This may open the way for new drugs that may help to treat these conditions.

The amounts of normal and compensatory polyamines of polyamine-requiring Escherichia coli mutants grown in the absence of polyamines were determined. Although aminopropylcadaverine, a compensatory polyamine, was synthesized by MA135 (speB) and DR112 (speA speB), no aminopropylcadaverine or only small amounts of aminopropylcadaverine were synthesized by EWH319 (speA speB speC speD) and MA261 (speB speC), respectively. The average mass doubling times of MA135, DR112, MA261, and EWH319 grown in the absence of polyamines were 113, 105, 260, and 318 min, respectively. The correlation of these values with the sum of spermidine plus aminopropylcadaverine suggested that aminopropylcadaverine is important for cell growth in the presence of limiting amounts of normal polyamines. This hypothesis is supported by the results of aminopropylcadaverine stimulation of the in vitro synthesis of polyphenylalanine and MS2 RNA replicase and of its stimulation of the growth of MA261. For the following reasons, it was concluded that aminopropylcadaverine was synthesized preferentially from cadaverine made by ornithine decarboxylase: aminopropylcadaverine was synthesized in relatively large amounts in cells (MA135 and DR112) which possess ornithine decarboxylase; ornithine decarboxylase catalyzed the decarboxylation of lysine in vitro, and the in vivo formation of aminopropylcadaverine was inhibited by an inhibitor of ornithine decarboxylase.

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.

Polyamines are essential for cell proliferation; therefore, we hypothesized that arginase I or arginase II activities, via production of ornithine for polyamine synthesis, may be limiting for proliferation of endothelial cells (EC). Bovine coronary venular EC stably transfected with a lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC) were utilized to test this hypothesis. Cell-proliferation assays showed that EC proliferation was markedly increased in AI-EC and AII-EC compared with lacZ-EC. Expression of proliferating cell nuclear antigen was also enhanced in AI-EC and AII-EC. DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to establish that increased polyamine synthesis was involved in mediating the enhanced growth of AI-EC and AII-EC. Addition of 5 mM DFMO to the culture medium completely abolished the differences in cellular putrescine concentrations and reduced the differences in spermidine concentrations among AI-EC, AII-EC, and lacZ-EC. The DFMO treatment also prevented an increase in AI-EC and AII-EC proliferation compared with lacZ-EC. Addition of 10 and 50 microM putrescine dose-dependently increased AI-EC, AII-EC, and lacZ-EC growth to the same extent. These results demonstrate that either arginase isoform can potentially play a role in modulating EC proliferation by regulating polyamine synthesis.