1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5'-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70-98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5'-Methylthioadenosine, 5'-ethylthioadenosine and 5'-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10-15mum and 50% inhibition of spermidine synthase at 30-45mum. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20mum respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5'-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient-dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.

l-Ornithine decarboxylase (ODC) is essential for polyamine synthesis and growth in mammalian cells; it provides putrescine that is usually converted into the higher polyamines, spermidine and spermine. Many highly specific and potent inhibitors of ODC are based on the lead compound alpha-difluoromethylornithine (DFMO), which is an enzyme-activated irreversible inhibitor. DFMO is accepted as a substrate by ODC and is decarboxylated, leading to the formation of a highly reactive species that forms a covalent adduct with either cysteine-360 (90%) or lysine-69 (10%). Both modifications inactivate the enzyme. ODC activity is normally very highly regulated at both transcriptional and post-transcriptional levels according to the growth state of the cell and the intracellular polyamine content. Experimental over-production of ODC can be caused by either transfection with plasmids containing the ODC cDNA with part of the 5'-untranslated region (5'UTR) deleted under the control of a very strong viral promoter, or transfection of plasmids that cause the overproduction of eIF-4E, reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5'UTR. In both cases, unregulated overexpression of ODC transforms NIH 3T3 cells to a neoplastic state. Along with studies showing that many tumor promoters increase ODC activity and that a number of preneoplastic conditions and tumor samples show high levels of ODC, these results suggest that ODC may act as an oncogene in an appropriate background. This provides a rationale for the possible use of ODC inhibitors as chemopreventive agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Dioecious white campion Silene latifolia has sex chromosomal sex determination, with homogametic (XX) females and heterogametic (XY) males. This species has become popular in studies of sex chromosome evolution. However, the lack of genes isolated from the X and Y chromosomes of this species is a major obstacle for such studies. Here, I report the isolation of a new sex-linked gene, Slss, with strong homology to spermidine synthase genes of other species. The new gene has homologous intact copies on the X and Y chromosomes (SlssX and SlssY, respectively). Synonymous divergence between the SlssX and SlssY genes is 4.7%, and nonsynonymous divergence is 1.4%. Isolation of a homologous gene from nondioecious S. vulgaris provided a root to the gene tree and allowed the estimation of the silent and replacement substitution rates along the SlssX and SlssY lineages. Interestingly, the Y-linked gene has higher synonymous and nonsynonymous substitution rates. The elevated synonymous rate in the SlssY gene, compared with SlssX, confirms our previous suggestion that the S. latifolia Y chromosome has a higher mutation rate, compared with the X chromosome. When differences in silent substitution rate are taken into account, the Y-linked gene still demonstrates significantly faster accumulation of nonsynonymous substitutions, which is consistent with the theoretical prediction of relaxed purifying selection in Y-linked genes, leading to the accumulation of nonsynonymous substitutions and genetic degeneration of the Y-linked genes.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.

Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.

Escherichia coli and Micrococcus luteus DNA topoisomerase I are found to promote catenation of double-stranded DNA rings. At low DNA concentration dimeric catenanes are the major catenated products; at high DNA concentration or when spermidine is present, catenanes containing more than two rings are formed. There is no requirement of extensive sequence homology between the conponent rings forming a catenane; dimeric catenanes between Pseudomonas phage PM2 DNA and E. coli plasmid pBR322 are readily formed. The formation of a dimeric catenane by these type I topoisomerases, however, requires the presence of at least one preexisting single-chain scission in one of the two component rings. This is in contrast to the cases with the type II DNA topoisomerases which can form catenanes made of covalently closed rings only. The catenanes formed by the type I enzymes can be unlinked by the same enzymes, or by DNA gyrase, a type II enzyme, upon dilution of the isolated catenanes. The catenation and decatenation of duplex DNA rings adds a fourth type of reaction promoted by these type I DNA topoisomerases to the three reported previously: relaxation of superhelical DNA, interconversion between single-stranded DNA rings with and without knots and the intertwining of single-stranded DNA rings of complementary sequences into a covalently closed duplex ring with a high linking number. All four topoisomerization reactions involve the crossing of one DNA strand through a transient break of another DNA strand. The new reaction reported here suggests that such a crossover event might not require pairing of complementary nucleotide sequences.

Electron microscopy is employed to examine DNA aggregates produced by three tripositively charged condensing agents. Spermidine, hexammine cobalt (III), and me8spermidine (in which the amine groups of spermidine are exhaustively methylated) all produce condensates. The predominant form of condensate observed is toroidal; however, me8spermidine produces a large fraction of rodlike condensates. Distributions of toroidal radii and estimated volumes suggest that the size of condensates depends on the condensing agent employed, its concentration, and the time elapsed after addition of condensing agent. While ligand charge seems to be the major factor in predicting condensing power, ligand structure influences the morphology and dimensions of the particles produced. The ability to form hydrogen bonds is not required to promote condensation, since me8spermidine has no NHs. There may be a kinetic barrier to condensation at low me8spermidine concentrations. The relative proportions of toroids and rods may depend on the energetic compensation between bending and binding in cyclic structures, or on rate-limiting formation of sharply bent or kinked regions in rods.

Trypanosoma brucei brucei S-adenosyl-L-methionine (AdoMet) decarboxylase was found to be relatively insensitive to activation by putrescine as compared with the mammalian enzyme, being stimulated by only 50% over a 10,000-fold range of putrescine concentrations. The enzyme was not stimulated by up to 10 mM-Mg2+. The Km for AdoMet was 30 microM, similar to that of other eukaryotic AdoMet decarboxylases. T.b. brucei AdoMet decarboxylase activity was apparently irreversibly inhibited in vitro by Berenil and reversibly by pentamidine and methylglyoxal bis(guanylhydrazone). Berenil also inhibited trypanosomal AdoMet decarboxylase by 70% within 4 h after administration to infected rats and markedly increased the concentration of putrescine in trypanosomes that were exposed to the drug in vivo. Spermidine and spermine blocked the curative effect of Berenil on model mouse T.b. brucei infections. This effect of the polyamines was probably not due to reversal of Berenil's inhibitory effects on the AdoMet decarboxylase.

Rapid, polyamine-induced degradation of mammalian ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC) is though to be controlled by the availability of a small, ODC-binding protein termed antizyme. In this study we have investigated the ability of antizyme to bind ODC protein in various altered physiological states. In particular, cold, NaCl, spermidine and deprivation of coenzyme and substrate enhance enzyme-antizyme complex formation and are all found to promote ODC homodimer dissociation. Conversely, conditions that maintain the active ODC homodimer state prevent antizyme binding and inactivation of ODC. Further, covalent modification of ODC near its active site by difluoromethylornithine or phosphate also increases its sensitivity to antizyme. These results suggest that the initial signal in ODC degradation may actually be a subtle conformational change in the enzyme that enables antizyme to bind to the enzyme and may subsequently facilitate its degradation.

Polyamine transport was examined in Chinese hamster ovary (CHO) cells because of the unique potential these cells hold for utilizing genetic approaches to study the mechanisms of polyamine transport, its regulation, and its function. Parental (control) CHO cells were shown to contain a polyamine transport system with characteristics consistent with polyamine-uptake properties described in other cell types. Polyamines appear to cross the plasma membrane via an energy-requiring transport system specific for putrescine, spermidine, spermine, and their analogues. A mutant line, CHOMG, selected for resistance to the toxicity of methylglyoxal bis(guanylhydrazone), was shown to lack a functional polyamine transport system. CHOMG cells provided the negative controls necessary to examine the role of polyamine transport in maintenance of intracellular polyamine levels and in the regulation of the polyamine metabolic enzymes. It was found that the repression of ornithine decarboxylase activity by polyamines and the induction of spermidine/spermine-N1 acetyltransferase by polyamine analogues including bis(ethyl)spermine derivatives required the presence of a functional polyamine transport system. The CHO-CHOMG model was also shown to provide a means for establishing the importance of the polyamine transport system in the toxicity of polyamine analogues. The inability of alpha-difluoromethylornithine-treated CHOMG cells to utilize extracellular polyamines to replenish depleted intracellular polyamine levels suggested a means by which polyamine transport-positive cells may be identified. Such a selection procedure will permit the use of CHOMG cells in the isolation of genes encoding proteins involved in polyamine transport.

Polyamines play essential functions in many aspects of cell biology. Plasma membrane transport systems for the specific uptake of polyamines exist in most eukaryotic cells but have been very recently identified at the molecular level only in the parasite Leishmania. We now report that the high affinity polyamine permease in Saccharomyces cerevisiae is identical to Agp2p, a member of the yeast amino acid transporter family that was previously identified as a carnitine transporter. Deletion of AGP2 dramatically reduces the initial velocity of spermidine and putrescine uptake and confers strong resistance to the toxicity of exogenous polyamines, and transformation with an AGP2 expression vector restored polyamine transport in agp2delta mutants. Yeast mutants deficient in polyamine biosynthesis required >10-fold higher concentrations of exogenous putrescine to restore cell proliferation upon deletion of the AGP2 gene. Disruption of END3, a gene required for an early step of endocytosis, increased the abundance of Agp2p, an effect that was paralleled by a marked up-regulation of spermidine transport velocity. Thus, AGP2 encodes the first eukaryotic permease that preferentially uses spermidine over putrescine as a high affinity substrate and plays a central role in the uptake of polyamines in yeast.

Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.

Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.

Nucleosomes are the basic elements of chromatin structure. Polyamines, such as spermine and spermidine, are small ubiquitous molecules absolutely required for cell growth. Photoaffinity polyamines bind to specific locations in nucleosomes and can change the helical twist of DNA in nucleosomes. Acetylation of polyamines reduces their affinity for DNA and nucleosomes, thus the helical twist of DNA in nucleosomes could be regulated by cells through acetylation. I suggest that histone and polyamine acetylation act synergistically to modulate chromatin structure. On naked DNA, the photoaffinity spermine bound preferentially to a specific 'TATA' sequence element, suggesting that polyamines may be involved in the unusual chromatin structure in this region. Further work is needed to test whether the specificities shown by photoaffinity polyamines are also shown by cellular polyamines; such experiments are now feasible.

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.

The localization and activity of polyamine oxidase (PAO; EC 1.5.3.3), was investigated in leaves and protoplasts of oat seedlings. Activity of the enzyme is highest with spermine as substrate; spermidine is also oxidized, but putrescine and cadaverine are unaffected by the enzyme. Protoplasts isolated following digestion of leaves with cellulase in hypertonic osmoticum showed no PAO activity, and about 80% of the total leaf PAO activity could be accounted for in the cell wall debris. Histochemical localization experiments showed intense PAO activity in guard cells and in vascular elements whose walls are not digested by cellulase. When protoplasts were cultured in a medium suitable for regeneration of cell wall, PAO activity could be detected as the cellulose wall developed. Thus, PAO appears to be localized in cell walls.Since applied spermine and spermidine prevent senescence of detached leaves, PAO activity was investigated during leaf senescence. The specific activity of PAO declines with increasing age of attached leaves and with increasing senescence of excised leaves incubated in darkness. This decline in enzyme activity, which parallels the decreases in chlorophyll and protein content used as measures of leaf senescence, suggests that the enzyme is not involved in the control of senescence of oat leaves.

The increased transcription and ultimate superinduction of the spermidine/spermine N(1)-acetyltransferase (SSAT) gene has been associated with the antineoplastic activity of several new antitumor polyamine analogues. In sensitive tumor cell types, the transcriptional induction appears to be regulated by the constitutive association of the transcription factor Nrf-2 with the recently discovered polyamine-responsive element. Using the yeast two-hybrid system, a new transcriptional cofactor, polyamine-modulated factor-1 (PMF-1), has been identified as a partner protein of Nrf-2 that, in combination with Nrf-2, regulates the polyamine analogue-induced transcription of SSAT. The human PMF-1 gene, located on chromosome 1 near the 1q12/1q21 border, yields an mRNA transcript of approximately 1.2 kilobases that codes for a 165-amino acid protein with a predicted molecular mass of approximately 20 kDa. The PMF-1 mRNA appears to increase in response to analogue exposure only in analogue-responsive cells. In addition to the transcriptional regulation of SSAT, PMF-1 or similar factors should be considered in the regulation of other polyamine-dependent genes.

Salt-tolerant Pokkali rice plants accumulate higher polyamines (PAs) such as spermidine (Spd) and spermine (Spm) in response to salinity stress, while the sensitive cultivarM-1-48 is unable to maintain high titres of these PAs under similar conditions. The effects of the triamine Spd and the tetramine Spm on physiological and biochemical changes in 12-day-old rice seedlings were investigated during salinity stress to determine whether they could protect the sensitive plants from stress effects. At physiological concentrations Spd and Spm significantly prevented the leakage of electrolytes and amino acids from roots and shoots induced by salinity stress. To different degrees they also prevented chlorophyll loss, inhibition of photochemical reactions of photosynthesis as well as downregulation of chloroplast-encoded genes like psbA, psbB, psbE and rbcL, indicating a positive correlation between salt tolerance and accumulation of higher PAs in rice. The inhibitory effect of salinity stress and its reversal by exogenous PAs were more pronounced in the salt-sensitiveM-1-48 plants than in the tolerant Pokkali plants.

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs.

Alcoholic beverage consumption is associated with an increased risk of upper gastrointestinal cancer. Acetaldehyde (AA), the first metabolite of ethanol, is a suspected human carcinogen, but the molecular mechanisms underlying AA carcinogenicity are unclear. In this work, we tested the hypothesis that polyamines could facilitate the formation of mutagenic alpha-methyl-gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (Cr-PdG) adducts from biologically relevant AA concentrations. We found that Cr-PdG adducts could be formed by reacting deoxyguanosine with muM concentrations of AA in the presence of spermidine, but not with either AA or spermidine alone. The identities of the Cr-PdG adducts were confirmed by both liquid and gas chromatography-mass spectrometry. Using a novel isotope-dilution liquid chromatography-mass spectrometry assay, we found that in the presence of 5 mM spermidine, AA concentrations of 100 microM and above resulted in the formation of Cr-PdG in genomic DNA. These AA levels are within the range that occurs in human saliva after alcoholic beverage consumption. We also showed that spermidine directly reacts with AA to generate crotonaldehyde (CrA), most likely via an enamine aldol condensation mechanism. We propose that AA derived from ethanol metabolism is converted to CrA by polyamines in dividing cells, forming Cr-PdG adducts, which may be responsible for the carcinogenicity of alcoholic beverage consumption.