The participation of polyamines and nonprotein sulfhydryls in the gastric cytoprotective mechanisms was studied using gastric mucosal lesions produced by acidified ethanol in rats as an experimental model. Treatment with prostaglandin E2 (PGE2), but not cimetidine, prevented the formation of gastric mucosal lesions. Oral administration of cadaverine, spermidine and spermine prevented the lesion formation by acidified ethanol in a dose-dependent manner. Indomethacin or acetazolamide had no influence on the cytoprotective effect of spermine, whereas sulfhydryl blockers such as iodoacetamide and N-ethylmaleimide partially blocked it. Sulfhydryl compounds such as cysteine, reduced glutathione (GSH), and cysteamine prevented the lesion formation induced by acidified ethanol. The concentration of nonprotein sulfhydryls in the gastric mucosa was significantly decreased at 1 hr after administration of acidified ethanol, and this decrease was partially prevented by spermine or PGE2. These results suggest that the cytoprotective effect of spermine may not be mediated by endogenous prostaglandins or alkaline secretion in the gastric mucosa, but may be partially related to endogenous sulfhydryl compounds.

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a transcription factor that plays an important role in regulating cell growth and differentiation. This protein plays a central role in lymphocyte and adipocyte differentiation and hepatic regeneration and in the control of inflammation and immunity in the liver and in cells of the myelomonocytic lineage. Our previous studies suggested that this protein could also have important functions in the brain. Therefore, we were interested in the identification of downstream targets of this transcription factor in cells of neural origin. We performed cDNA microarray analysis and found that a total of 48 genes were up-regulated in C/EBPbeta-overexpressing neuronal cells. Of the genes that displayed significant changes in expression, several were involved in inflammatory processes and brain injury. Northern blot analysis confirmed the up-regulation of ornithine decarboxylase, 24p3/LCN2, GRO1/KC, spermidine/spermine N(1)-acetyltransferase, xanthine dehydrogenase, histidine decarboxylase, decorin, and TM4SF1/L6. Using promoter-luciferase reporter transfection assays, we showed the ornithine decarboxylase and 24p3 genes to be biological downstream targets of C/EBPbeta in neuroblastoma cells. Moreover, the levels of C/EBPbeta protein were significantly induced after neuronal injury, which was accompanied by increased levels of cyclooxygenase-2 enzyme. This strongly supports the concept that C/EBPbeta may play an important role in brain injury.

Synthesis of S-adenosylmethionine decarboxylase (AdoMetDC), a key regulated enzyme in the pathway of polyamine biosynthesis, is feedback-controlled at the level of translation by spermidine and spermine. The peptide product of an upstream open reading frame (uORF) in the mRNA is solely responsible for polyamine regulation of AdoMetDC translation. Using a primer extension inhibition assay and in vitro protein synthesis reactions, we found ribosomes paused at or close to the termination codon of the uORF. This pause was greatly diminished with the altered uORFs' sequences that abolish uORF regulation in vivo. The half-life of the ribosome pause was related to the concentration of polyamines present but was unaffected by magnesium concentration. Furthermore, inhibition of translation initiation at a reporter gene placed downstream of the AdoMetDC uORF directly correlated with the stability of the ribosome pause at the uORF. These observations are consistent with a model in which regulation of ribosome pausing at the uORF by polyamines controls ribosome access to the downstream AdoMetDC reading frame.

Ferroptosis is a form of lipid peroxidation-induced cell death that can be regulated in many ways, from altering the activity of antioxidant enzymes to the level of transcription factors. The p53 tumor suppressor is 'the guardian of the genome' that participates in the control of cell survival and division under various stresses. Beyond its effects on apoptosis, autophagy, and cell cycle, p53 also regulates ferroptosis either through a transcriptional or posttranslational mechanism. On one hand, p53 can enhance ferroptosis by inhibiting the expression of SLC7A11 (solute carrier family 7 member 11) or by enhancing that of SAT1 (spermidine/spermine N1-acetyltransferase 1) and GLS2 (glutaminase 2). On the other hand, p53 suppresses ferroptosis through the direct inhibition of DPP4 (dipeptidyl peptidase 4) activity or by the induction of CDKN1A/p21 (cyclin dependent kinase inhibitor 1 A) expression. Here, we review recent discoveries and emerging trends in the study of the ferroptosis network and highlight the context-dependent impact of p53 on ferroptosis and oxidative stress.

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells.

The polyamines are abundant biogenic cations implicated in many biological processes. Despite a plethora of evidence on polyamine-induced DNA conformational changes, no thorough study of their effects on the activities of sequence-specific DNA binding proteins has been performed. We describe the in vitro effects of polyamines on the activities of purified, representative DNA-binding proteins, and on complex protein mixtures. Polyamines at physiological concentrations enhance the binding of several proteins to DNA (e.g. USF, TFE3, Ig/EBP, NF-IL6, YY1 and ICP-4, a herpes simplex virus gene regulator), but inhibit others (e.g. Oct-1). The degree of enhancement correlates with cationic charge; divalent putrescine is ineffective whereas tetravalent spermine is more potent than trivalent spermidine. Polyamine effects on USF and ICP-4 result from increased rate of complex formation rather than a decreased rate of dissociation. DNAse I footprint analysis indicated that polyamines do not alter DNA-protein contacts. Polyamines also facilitate formation of complexes involving binding of more than one protein on a DNA fragment.

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.

Over the last few years a remarkable progress has been made in the understanding of parasites biochemistry, molecular biology, and immunology. This progress is especially encouraging in that emphasis on drug development is shifting from random screening towards a more rational approach. A number of peculiar aspects characteristic of parasites which are not present in other organisms and that might be exploitable for the design of specific agents have been described recently. One of these aspects is their deficiency in defense mechanisms against oxygen toxicity. Catalase is absent in many parasites. Distinct superoxide dismutases have been detected and specific inhibitors of these enzymes have been investigated. Glutathione is absent in some anaerobic protozoa. Peroxidase and reductase activities dependent on a glutathione-spermidine cofactor termed trypanothione have been detected in several trypanosomatids and apparently replace the glutathione peroxidase-glutathione reductase system of other eukaryotic cells. Free radical intermediates have been shown to be involved in the reaction of enzymes present in anaerobic protozoa. In addition, a number of antiparasitic agents have been shown to exert their actions through a free radical metabolism: nitro compounds used against trypanosomatids, anaerobic protozoa and helminths; crystal violet used in blood banks to prevent blood transmission of Chagas' disease; the antimalarial primaquine, chloroquinine, and quinhasou; and quinones active in vitro and in vivo against different parasites.

Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one-third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants.

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.

This investigation shows whether polyamines and ornithine decarboxylase have a role in duodenal mucosal repair following stress-induced microscopic damage. Rats were fasted for 22 hours, placed in restraint cages, and immersed in water to the xiphoid process for 6 hours. Animals were killed either immediately after the period of stress or at 2-hour intervals up to 24 hours thereafter. Duodenal mucosa was examined histologically, and ornithine decarboxylase and polyamine levels were measured. Ornithine decarboxylase activity was increased significantly up to 6 hours following stress, peaking at 4 hours at a level 10 times the prestress control. By 8 hours, enzyme activity had returned to near normal. Increases in mucosal putrescine, spermidine, and spermine content paralleled the changes in ornithine decarboxylase activity and peaked 4 hours after stress. Stress resulted in microscopic damage evidenced by a nearly complete absence of villi. Significant macroscopic lesions were not present following stress. Mucosal repair was evident 12 hours after stress and almost complete by 24 hours, although the restituted villi were short and blunted. The decreases in mucosal DNA, RNA, and protein content caused by stress were restored and reached near-normal levels 12 hours after the period of stress. In animals given the specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, increases in duodenal mucosal ornithine decarboxylase activity and polyamine levels were inhibited and mucosal repair was almost completely prevented following stress. alpha-Difluoromethylornithine also prevented the recovery of DNA, RNA, and protein content of the duodenal mucosa. These results indicate that duodenal mucosal damage following stress is repaired rapidly; the repair process is accompanied by significant increases in ornithine decarboxylase activity and polyamine levels; and the increases in ornithine decarboxylase and polyamines are absolutely required for the normal repair of the mucosa.

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

We have measured forces generated by multivalent cation-induced DNA condensation using single-molecule magnetic tweezers. In the presence of cobalt hexammine, <em>spermidine</em>, or spermine, stretched DNA exhibits an abrupt configurational change from extended to condensed. This occurs at a well-defined condensation force that is nearly equal to the condensation free energy per unit length. The multivalent cation concentration dependence for this condensation force gives the apparent number of multivalent cations that bind DNA upon condensation. The measurements show that the lower critical concentration for cobalt hexammine as compared to <em>spermidine</em> is due to a difference in ion binding, not a difference in the electrostatic energy of the condensed state as previously thought. We also show that the resolubilization of condensed DNA can be described using a traditional Manning-Oosawa cation adsorption model, provided that cation-anion pairing at high electrolyte concentrations is taken into account. Neither overcharging nor significant alterations in the condensed state are required to describe the resolubilization of condensed DNA. The same model also describes the <em>spermidine</em>3+/Na+ phase diagram measured previously.

Complete inhibition of polyamine catabolism is possible by combined administration of two compounds. Aminoguanidine (25 mg/kg body wt., intraperitoneally) inhibits all reactions that are catalysed by copper-containing amine oxidases (CuAO). The products of the CuAO-catalysed reactions cannot be reconverted into polyamines (terminal catabolism) and therefore usually escape observation. N1-Methyl-N2-(buta-2,3-dienyl)butane-1,4-diamine (MDL 72521) is a new inhibitor of polyamine oxidase. It inhibits completely the degradation of N1-acetylspermidine and N1-acetylspermine. The enhanced excretion of N1-acetylspermidine in urine after administration of 20 mg of MDL 72521/day per kg body wt. is a measure of the rate of spermidine degradation in vivo to putrescine, and thus of the quantitative significance of the interconversion pathway. From the enhancement of total polyamine excretion by aminoguanidine-treated rats, one can calculate that only about 40% of the polyamines that are destined for elimination are usually observed in the urine, the other 60% being catabolized along the CuAO-catalysed pathways. The normally observed urinary polyamine pattern gives, therefore, an unsatisfactory picture of the actual polyamine elimination. Although aminoguanidine alone is sufficient to block terminal polyamine catabolism, rats that were treated with a combination of aminoguanidine and MDL 72521 excrete more polyamines than those that received aminoguanidine alone. The reason is that a certain proportion of putrescine, which is formed by degradation of spermidine, is normally reutilized for polyamine biosynthesis. In MDL 72521-treated animals this proportion appears in the urine in the form of N1-acetylspermidine. Thus it is possible to determine polyamine interconversion and re-utilization in vivo and to establish a polyamine balance in intact rats by using specific inhibitors of the CuAO and of polyamine oxidase.

Rapid coagulation of seminal fluid in rats, guinea pigs, and several other mammalian species including certain non-human primates is responsible for the post-coital formation of copulatory plugs in the vagina. The clotting of rodent seminal plasma results from coagulation of certain proteins derived from the seminal vesicles by enzymes secreted mainly by the coagulating (anterior prostate) gland. Several lines of evidence indicate that the clotting enzymes of coagulating gland secretions are transglutaminases, and that the extreme insolubility of the seminal clot in rodents is due to transglutaminase-catalyzed formation of epsilon(gamma-glutamyl)lysine cross-links between polypeptide chains. Various features of the apparently unique forms of transglutaminases produced by rat coagulating gland and the actions of these enzymes on vesicular secretory and other proteins are discussed. The aliphatic polyamines spermidine and spermine are incorporated covalently into the proteins of the clot as the corresponding N-mono-epsilon-(gamma-glutamyl)- and N,N-bis(gamma-glutamyl)-adducts during the enzymatic coagulation process. At the greater than millimolar concentrations at which cross-spermidine and spermine are present in normal rat seminal plasma, these polyamines attenuate the formation of hard clots in reconstituted rat semen coagulation systems, seemingly by competing with lysyl residues in vesicular secretion proteins as transglutaminase amine donor substrates, and thus preventing formation of epsilon-(gamma-glutamyl)lysine cross-bridges. It is proposed that in those species such as the rat and man in which seminal plasma contains large amounts of spermidine and(or) spermine of prostatic origin, the seminal polyamines may serve to stop blockage of the urethra by preventing too explosive a rate of seminal clot formation during the ejaculatory process.

Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis.

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.

Endogenous polyamines such as spermine and spermidine have multiple effects in the central nervous system and have been suggested to be neurotransmitters or neuromodulators. One effect of the polyamines is to regulate the activity of the N-methyl-D-aspartate receptor (NMDAR) channel subtype of glutamate receptor channels. The effects of polyamines on NMDAR currents are complex, suggesting the presence of one or more polyamine-binding sites on the receptor channel. Electrophysiological studies have shown that polyamines enhance NMDAR currents by increasing channel opening frequency and by increasing the affinity of the receptor for glycine. Polyamines have been shown to reduce NMDAR currents by producing voltage-dependent reduction of single-channel amplitudes and/or by producing an open channel block. Recent molecular biological studies have shown that the polyamine effects on NMDAR channels involve interactions with multiple NMDAR subunits and are characterizing the structural basis for the polyamine regulation of NMDAT receptor channels.

The binding characteristics of [3H]gabapentin, the radiolabelled analogue of the novel anticonvulsant gabapentin (1-(aminomethyl)cyclohexaneacetic acid) were studied using purified synaptic plasma membranes prepared from rat cerebral cortex. In 10 mM HEPES buffer [3H]gabapentin bound to a single population of sites with high affinity (KD = 38 +/- 2.8 nM) with a maximum binding capacity of 4.6 +/- 0.4 pmol/mg protein, reaching equilibrium after 30 min at 20 degrees C. This novel site was unique to the central nervous system with little or no specific [3H]gabapentin being measurable in a range of peripheral tissues. Binding was potently inhibited by a range of gabapentin analogues and 3-alkyl substituted gamma-aminobutyric acid (GABA) derivates although GABA itself and the selective GABAB receptor ligand baclofen, were only weakly active. Gabapentin itself (IC50 = 80 nM) and 3-isobutyl GABA (IC50 = 80 nM) which also has anticonvulsant properties, showed the highest affinity for the binding site. Of a wide range of other pharmacologically active compounds only the polyamines spermine and spermidine influenced [3H]gabapentin binding, with both compounds producing a maximum of 50% inhibition of specific binding. Magnesium ions produced a similar pattern of inhibition but the effect of the polyamines and magnesium ions were not additive. The data provide evidence for the existence in brain of a novel binding site that may mediate the anticonvulsant effects of gabapentin and other potential anticonvulsant compounds.

Polyamine biosynthesis is an ancient metabolic pathway present in all organisms. Aminopropyltransferases are key enzymes that mediate the synthesis of spermidine, spermine, and thermospermine. The relatively high sequence similarity between aminopropyltransferases and their similarity with putrescine N-methyltransferases (PMT) raises the question of whether they share a common ancestor or have evolved by convergence. Here we show that aminopropyltransferases and PMT are phylogenetically interconnected, and the different activities have been generated by unusually frequent events of diversification of existing functions. Although all spermidine synthases (SPDSs) derive from a common ancestor preceding the separation between prokaryotes and eukaryotes, they have been the origin of a variety of new activities. Among those, spermine synthases (SPMSs) represent a novelty independently arisen at least 3 times, in animals, fungi, and plants. The most parsimonious mechanism would involve the duplication and change of function of preexisting SPDS genes in each phylum. Although spermine is not essential for life, the repeated invention of SPMS and its conservation strongly argues for an evolutionary advantage derived from its presence. Moreover, the appearance of thermospermine synthase (tSPMS) in several genera of Archaea and Bacteria was accompanied by a loss of SPDS, suggesting that the new activity originated as a change of function of this enzyme. Surprisingly, tSPMS was later acquired by plants at an early stage of evolution by horizontal gene transfer and has proven to be essential for vascular development in tracheophytes. Finally, the synthesis of nicotine and tropane alkaloids in Solanales was favored by the origination of a new activity, PMT, as a duplication and change of function from SPDS.

Recently, iron acquisition and, more specifically, enzymes involved in siderophore biosynthesis have become attractive targets for discovery of new antibiotics. Accordingly, targeted inhibition of the biosynthesis of petrobactin, a virulence-associated siderophore encoded by the asb locus in Bacillus anthracis, may hold promise as a potential therapy against anthrax. This study describes the biochemical characterization of AsbC, the first reported 3,4-dihydroxybenzoic acid-AMP ligase, and a key component in the biosynthesis of DHB-spermidine (DHB-SP), the first isolable intermediate in petrobactin biosynthesis. AsbC catalyzes adenylation to the corresponding AMP ester of the unusual precursor 3,4-dihydroxybenzoate, in addition to benzoate substrates bearing hydrogen bond-donating substituents at the para and meta positions on the phenyl ring. In a second reaction, AsbC catalyzes transfer of the activated starter unit to AsbD, an aryl carrier protein similar to acyl and peptidyl carrier proteins that function in fatty acid, polyketide, and nonribosomal peptide biosynthesis. A third protein, AsbE, is shown to be responsible for condensation of 3,4-dihydroxybenzoyl-AsbD with spermidine, providing the DHB-spermidine arms that are linked to citrate for assembly of petrobactin. On the basis of the selective substrate profile of AsbC, a nonhydrolyzable analogue of 3,4-DHB-AMP was synthesized and shown to effectively inhibit AsbC function in vitro.

OBJECTIVE

Global DNA hypomethylation in rheumatoid arthritis synovial fibroblasts (RASFs) contributes to their intrinsic activation. The aim of this study was to investigate whether increased polyamine metabolism is associated with a decreased level of S-adenosyl methionine (SAM), causing global DNA hypomethylation.

METHODS

Synovial fibroblasts were isolated from synovial tissue obtained from 12 patients with RA and from 6 patients with osteoarthritis (OA). The cells were stained for S-adenosyl methionine decarboxylase (AMD), spermidine/spermine N1-acetyltransferase (SSAT1), polyamine-modulated factor 1-binding protein 1 (PMFBP1), solute carrier family 3 member 2 (SLC3A2), DNA methyltransferase 1 (DNMT-1), α9 integrin, and β1 integrin and analyzed by flow cytometry. Nuclear 5-methylcytosine (5-MeC) was measured by flow cytometry, the expression of diacetylspermine (DASp) in cell culture supernatants and cell extracts was determined by enzyme-linked immunosorbent assay, and SAM expression in cell extracts was measured by fluorometry.

RESULTS

The expression of SSAT1, AMD, and PMFBP1 was significantly increased in RASFs compared with OASFs. The expression of DASp in cell culture supernatants and the expression of SLC3A2 were significantly elevated in RASFs. The levels of SAM in cell culture extracts, as well as the levels of DNMT-1 protein and 5-MeC, were significantly reduced in RASFs. Parameters of polyamine metabolism were negatively correlated with the expression of SAM, DNMT-1, and 5-MeC.

CONCLUSIONS

These data clearly show that intrinsic elevations of PMFBP1 and SSAT1 enhance the catabolism and recycling of polyamines in RASFs and suggest that high consumption of SAM via this pathway is an important factor contributing to global DNA hypomethylation in these cells.

The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.