When mammalian cells are grown in medium containing [3H]spermidine, a single major tritiated protein identical to eukaryotic initiation factor 4D becomes labeled. This protein contains 1 residue/molecule of tritiated hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), a rare amino acid which has been found in no other protein. In order to investigate the conservation of this protein, we examined two nonmammalian eukaryotes, the yeast Saccharomyces cerevisiae and the insect Drosophila melanogaster, and the eubacterial prokaryote Escherichia coli for the presence of the hypusine-containing protein. When the eukaryotic cells were grown in the presence of [3H]spermidine, electrophoretic analysis revealed a single labeled protein. In each case, the apparent molecular weight was near 18,000 and the relative pI was approximately 5.2, similar to the hypusine-containing protein of mammals. Amino acid analysis confirmed the presence of tritiated hypusine in each case, and silver staining of two-dimensional polyacrylamide gels demonstrated that, in yeast and fruit flies as in mammals, the protein is relatively abundant. In the eubacterium E. coli, one tritiated protein was predominant, but its molecular weight was 24,000 and we found no evidence that it contained tritiated hypusine. We found no evidence for the existence of the hypusine-containing protein in the archaebacterium Methanococcus voltae. These data suggest that the hypusine-containing protein is conserved among eukaryotes.

Putrescine and spermidine accumulate in cereal cells and protoplasts exposed to various osmotica (sorbitol, mannitol, proline, betaine, or sucrose). The response is fast (1-2 hour lag), massive (50- to 60-fold increase in putrescine), and is not due to release of putrescine from a bound form or to conversion from spermidine. It rather involves the activation of the biosynthetic pathway mediated by arginine decarboxylase (ADC; EC 4.1.1.19) (Flores and Galston 1982 Science 217: 1259). Polyamine accumulation and the rise in ADC activity in osmotically stressed tissue are prevented by ADC inhibitors (alpha-difluoromethylarginine, d-arginine, and l-canavanine) but are not affected by alpha-difluoromethylornithine and methylornithine, inhibitors of the alternative putrescine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17). Putrescine accumulation by oat and corn leaves is maximal in solutions only slightly hyperosmotic (0.4 molar). The stress response, which declines with leaf age, is completely prevented by cycloheximide (10 to 50 micrograms per milliliter) when added during the first hour of exposure to osmoticum, and partially by transcription inhibitors (cordycepin, Actinomycin D, 5 to 20 micrograms per milliliter). Oat seedlings allowed to wilt by withholding water also show a rise in polyamine titer and ADC activity. This response is not readily reversible upon rewatering.

Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke.

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA(3)), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA(3), ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA(3) were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 microg ml(-1)). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 microg ml(-1)). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus L-methionine (3.94 ng ml(-1) h(-1)) and Az39 cultured in NFb plus L-lysine (36.55 ng ml(-1) h(-1)). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.

Saccharomyces boulardii is a yeast widely used in humans for the prevention and treatment of infectious enteritis and Clostridium difficile-associated enterocolopathies. After oral administration to human volunteers or growing rats, S. boulardii enhances markedly the expression of intestinal enzymes as well as the production of the polymeric immunoglobulin receptor by mechanisms that remain unknown. We have analyzed the role of the yeast polyamines as potential mediators in the intestinal trophic response. In weanling rats (d 20 to d 30), a daily dose of 100 mg of lyophilized S. boulardii produced significant (p < 0.025) increases in sucrase (157%) and maltase (47%) activities. This dose corresponded to a total oral load of 678 nmol of polyamines per day (spermidine; 376 +/- 32, spermine: 293 +/- 26, putrescine: 9.5 +/- 1.4 nmol/100 mg). Spermine, given orally to growing rats at doses nearly equivalent (500 nmol) to the load of polyamines provided by the yeast (678 nmol), reproduced similar enzymatic changes, including a 2.5-fold induction of sucrase, and enhanced maltase activity (+24%). Spermidine and spermine concentrations measured in the jejunal mucosa of treated rats were increased over matched controls by 21.4% (p < 0.005) and 21.9%, respectively (p < 0.002). After being centrifuged and filtered to discard residual yeast cells, 2-mL samples of jejunal and ileal fluid collected from S. boulardii-treated rats by intestinal flushing contained higher levels of spermidine (48 and 60%) and spermine (150 and 316%) than did control rats. Our data indicate that lyophilized S. boulardii exerts trophic effects on the small intestine that are likely mediated by the endoluminal release of spermine and spermidine.

The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post-infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell-wall breaking enzymes (muramidases). While a low-level bacteriolysis sometimes occurs physiologically, due to "mistakes" in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta-lactam antibiotics or by a large variety of bacteriolysis-inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram-positive bacteria and phospholipids in Gram-negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell-wall- and -membrane-associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta-lactams, could be effectively inhibited by sulfated polysaccharides, by D-amino acids as well as by certain anti-bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non-biodegradable cell-wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor-cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti-inflammatory agents. These might inhibit the synergistic tissue- and- organ-damaging "cross talks" which lead to septic shock and to additional post-infectious sequelae.

Polyamine catabolism is rate limited by spermidine/spermine N1-acetyltransferase (SSAT). Although the amino acid sequence of SSAT is known, the substrate binding and catalytic sites are not. The goal of this study was to define the region responsible for acetyl coenzyme A binding. Human SSAT contains a region of 20 amino acids homologous to several microbial antibiotic N-acetyltransferases. The highest homology is represented in the Campylobacter coli streptothricin acetyltransferase sat4 gene, where 16 identical or highly conserved amino acids exist in a 20-residue stretch. The most conserved residues within this region are RGFGIGS beginning at Arg-101 in the human SSAT. Site-directed mutations to Arg-101, Gly-104, and Gly-106 resulted in proteins with no measurable activity. The G102D mutation produced a partially active protein with a decreased affinity for acetyl coenzyme A and with a Km >10-fold that of the wild-type protein. Analysis using the PredictProtein program suggests a common structure among the microbial and eukaryotic N-acetyltransferases in the region corresponding to the RGFGIGS of human SSAT consisting of an alpha-helix usually preceded by a glycine loop. Our data are consistent with the hypothesis that Arg-101 and the proximal glycine loop are necessary for the activity of human SSAT.

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 microM increase F(V) as efficiently as 100 microM of spermidine or 1000 microM of putrescine or 1000 microM of Mg(2+). It is also demonstrated that the increase in F(V) derives mainly from the contribution of PSIIalpha centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.

Sequence analysis of the chromosomal Tn5lacZ flanking regions of the Pseudomonas fluorescens WCS365 competitive root colonization mutant PCL1206 showed that the Tn5lacZ is inserted between genes homologous to bioA and potF. The latter gene is the first gene of the potF1F2GHI operon, which codes for a putrescine transport system in Escherichia coli. The position of the Tn5lacZ suggests an effect on the expression of the pot operon. A mutation in the potF1 gene as constructed in PCL1270, however, had no effect on competitive root colonization. The rate of uptake of [1,4-14C]putrescine by cells of mutant PCL1206 appeared to be increased, whereas cells of strain PCL1270 were strongly impaired in the uptake of putrescine. Dansylation of tomato root exudate and subsequent thin-layer chromatography showed the presence of a component with the same Rf value as dansyl-putrescine, which was identified as dansyl-putrescine by mass spectrometric analyses. Other polyamines such as spermine and spermidine were not detected in the root exudate. Growth of mutant strains, either alone or in competition with the wild type, was tested in media containing putrescine, spermine, or spermidine as the sole nitrogen source. The results show that mutant PCL1206 is strongly impaired in growth on putrescine and slightly impaired on spermine and spermidine. The presence of the polyamines had a similar effect on the growth rate of strain PCL1270 in the presence of putrescine but a less severe effect in the presence of spermine and spermidine. We conclude that an increased rate of putrescine uptake has a bacteriostatic effect on Pseudomonas spp. cells. We have shown that putrescine is an important tomato root exudate component and that root-colonizing pseudomonads must carefully regulate their rate of uptake because increased uptake causes a decreased growth rate and, therefore, a decreased competitive colonization ability.

Polyamine oxidase (PAO) is a FAD-dependent enzyme with a molecular mass of about 62 kDa, present with high activity in most tissues of vertebrates. Structural requirements of a substrate for PAO are two positively charged amino groups, separated by a short carbon chain and an alkyl substituent on one or both nitrogen atoms. Spermine and the monoacetyl derivatives N1-acetylspermine and N1-acetylspermidine appear to be the natural substrates. Spermidine is only poorly oxidized by PAO. Using O2, the substrates are oxidatively cleaved by PAO to form equimolar amounts of an amine, an aldehyde and hydrogen peroxide. PAO is an integral part of the polyamine interconversion cycle, a major intracellular regulatory system, which contributes to the maintenance of polyamine homeostasis in non-proliferating cells, including brain cells. Selective inactivators were used as tools in the elucidation of the functions of PAO. Interestingly, even long-term inactivation of PAO did not provoke behavioral changes in experimental animals, despite considerable changes in polyamine metabolism. PAO inactivation, however, improves the growth-inhibitory effects of inhibitors of polyamine biosynthetic enzymes and the antitumoral effects of some structural analogs of the polyamines.

Because the excitable properties of neurons in the neocortex depend on the characteristics of voltage-gated Na(+) channels, factors which regulate those characteristics can fundamentally modify the dynamics of cortical circuits. Here, we report on a novel neuromodulatory mechanism that links the availability of Na(+) channels to metabolism of polyamines (PAs) in the cerebral cortex. Using single channel and whole-cell recordings, we found that products of PA metabolism, the ubiquitous aliphatic polycations spermine and spermidine, are endogenous blockers of Na(+) channels in layer 5 pyramidal cells. Because the blockade is activity-dependent, it is particularly effective against Na(+) channels which fail to inactivate rapidly and thus underlie the persistent Na(+) current. At the level of the local cortical circuit, pharmacological depletion of PAs led to increased spontaneous spiking and periods of hypersynchronous discharge. Our data suggest that changes in PA levels, whether associated with normal brain states or pathological conditions, profoundly modify Na(+) channel availability and thereby shape the integrative behavior of single neurons and neocortical circuits.

Selective cleavage of phosphodiester bonds in RNA is important in the processing of large RNA molecules. This paper reports specific cleavage at UA sequences in single stranded oligoribonucleotides as short as hexamers. The hydrolysis between U and A leaves a 2',3'-cyclic phosphate on the 5'-side and a 5'-hydroxyl group on the 3' side of the cleavage. The hydrolysis is promoted by a wide range of cofactors, including polymeric organic compounds such as polyvinylpyrrolydone (PVP) and by proteins. A variety of experiments suggests the cleavage is not due to contamination by ribonuclease. The rate of cleavage is a function of oligoribonucleotide, PVP and spermidine concentrations. Mg2+ is not required. The phenomenon described here can potentially provide a relatively simple way of coding chemical stability into single stranded RNA based on its sequence and structure. This process seems to be similar to that involved in post-transcriptional degradation of mRNA.

We have investigated the salt sensitivity of the hexagonal-to-cholesteric phase transition of spermidine-condensed DNA. This transition precedes the resolubilization of precipitated DNA that occurs at high spermidine concentration. The sensitivity of the critical spermidine concentration at the transition point to the anion species and the NaCl concentration indicates that ion pairing of this trivalent ion underlies this unusual transition. Osmotic pressure measurements of spermidine salt solutions are consistent with this interpretation. Spermidine salts are not fully dissociated at higher concentrations. The competition for DNA binding among the fully charged trivalent ion and the lesser charged complex species at higher concentrations significantly weakens attraction between DNA helices in the condensed state. This is contrary to the suggestion that the binding of spermidine at higher concentrations causes DNA overcharging and consequent electrostatic repulsion.

The influence of cations, temperature, and stem length on the supercoil-induced transition from the linear form to the cruciform state at certain inverted repeats of pVH51 and pBR322 was investigated. In general, conditions which stabilize duplex DNA over single-stranded DNA shifted the transition to higher negative superhelical density values due to an increase in the unfavorable free energy of cruciform formation. Specifically, increasing sodium or magnesium ion concentrations brought about a corresponding increase in the negative superhelical density required to cause cruciform formation at the major inverted repeat of both plasmids. A notable exception was the inverted repeat found in both of these plasmids (at position 1009 of pVH51 and 3123 of pBR322) for which Mg(II) concentrations between 1 and 5 mM brought about a lowering of the negative supercoiling required to cause cruciform extrusion at this site, suggesting a specific complex between the cruciform and magnesium. Increasing temperatures from 15 up to 45 degrees C for the pVH51 major inverted repeat and 37 degrees C for that of pBR322 shifted the transition to lower negative superhelical densities. Further increases brought about a shift to higher negative densities. For the two inverted repeats examined within pVH51, various divalent metal ions and spermidine resulted in the following hierarchy: Mn(II) less than Zn(II) less than Mg(II) less than Co(II) less than spermidine, where the transition midpoint was at lowest negative density values for Mn(II) and highest for spermidine. This hierarchy agrees qualitatively with the relative affinity of the cations for DNA-phosphates versus the bases. The influence of stem length on the supercoil-induced transition to the cruciform state was studied by in vitro deletion of portions of the pVH51 major inverted repeat. Decreasing the stem length from 13 to 10 base pairs (bp) had no effect on the ability of this sequence to adopt the cruciform state. However, a further reduction of 3 bp to give a stem length of 7 bp completely abolished the ability of this region of DNA to exist in the cruciform state, at least up to a density of -0.15. Thus, a very sharp dependency on stem length exists for cruciform formation within an inverted repeat region possessing a potential loop of five nucleotides.

Purified recA protein, which is essential for genetic recombination of Escherichia coli, catalyzed ATP-dependent homologous pairing of double-stranded DNA and single-stranded fragments to form D-loops. When the double-stranded DNA was nicked circular DNA (form II) or linear DNA (form III), the reaction proceeded nearly linearly during 30 min of incubation at 37 degrees C. When the double-stranded DNA was superhelical (form I), anomalous kinetics was observed. This anomaly was suppressed by the addition of spermidine without affecting the final yield of D-loops. The formation of D-loops required stoichiometric amounts of recA protein, which were proportional to the concentration of single-stranded DNA but which were not affected by the concentration of double-stranded DNA. With form II or III DNA as the recipient for the formation of D-loops, the rate of the reaction was greatest when there was one monomer of recA protein/2-3 nucleotide residues of single-stranded DNA; larger amounts of single-stranded DNA inhibited the reaction. The formation of D-loops was half inhibited by 30 mM NaCl and by 0.6 mM ADP, one of the products of the reaction. The thermal stability of D-loops made by recA protein was the same as that of D-loops made by annealing. In addition to pairing linear single strands with duplex DNA, recA protein made joint molecules from single-stranded circular DNA and homologous form II or III DNA. According to these and previous observations (Cunningham, R. P., DasGupta, C., Shibata, T., and Radding, C. M. (1980) Cell 20, 223-235), rcA protein will stably pair two molecules of DNA if one of them is single-stranded or partially single-stranded and if either molecule has a free end.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.

An exonuclease activity copurified with herpes simplex virus type I (HSV-1) DNA polymerase through DNA-cellulose column chromatography and comigrated with DNA polymerase activity on nondenaturing gel electrophoresis at varied polyacrylamide concentrations. A gapped duplex DNA was the preferred substrate for this exonuclease activity since the hydrolytic activity on this type of DNA was much greater than the hydrolysis of either native or heat-denatured DNA. Using 3'-terminally labeled activated calf thymus DNA as substrate, the exonuclease activity was found to be activated by salt and spermidine in a manner identical with HSV-1 DNA polymerase. This activation was accompanied by increases in apparent Km and Vmax values of the activated DNA substrate. Phosphonoformic acid inhibited both DNA polymerase and exonuclease activities uncompetitively with respect to activated DNA and had a Ki of 2.4 microM at an ionic strength of 0.25 mu. Of the nucleoside 5'-monophosphates tested only the purine ribonucleotides inhibited the exonuclease activity. The inhibition was noncompetitive with respect to DNA, and GMP was about twice as potent as AMP or IMP. 9-beta-D-arabinosyladenine 5'-monophosphate (araAMP) could be incorporated into DNA by HSV-1 DNA polymerase; however, 9-beta-D-arabinosyladenine 5'-triphosphate would not replace dATP in supporting in vitro HSV-1 DNA synthesis. AraAMP incorporated into primer termini caused a significant decrease in the rate of subsequent primer elongation. These 3'-terminal araAMP residues could be removed by the HSV-1 DNA polymerase-associated exonuclease activity in a manner dependent on GMP concentration.

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent Km=89 microM, Ki(s)=1mM, k(cat)=2s-1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent Km values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 microM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.

Two different approaches were used to examine the in vivo role of polyamines in causing inward rectification of potassium channels. In two-microelectrode voltage-clamp experiments, 24-hr incubation of Xenopus oocytes injected with 50 nl of difluoromethylornithine (5 mM) and methylglyoxal bis(guanylhydrazone) (1 mM) caused an approximate doubling of expressed Kir2.1 currents and relieved rectification by causing an approximately +10-mV shift of the voltage at which currents are half-maximally inhibited. Second, a putrescine auxotrophic, ornithine decarboxylase-deficient Chinese hamster ovary (O-CHO) cell line was stably transfected with the cDNA encoding Kir2.3. Withdrawal of putrescine from the medium led to rapid (1-day) loss of the instantaneous phase of Kir2.3 channel activation, consistent with a decline of intracellular putrescine levels. Four days after putrescine withdrawal, macroscopic conductance, assessed using an 86Rb+ flux assay, was approximately doubled, and this corresponded to a +30-mV shift of V1/2 of rectification. With increasing time after putrescine withdrawal, there was an increase in the slowest phase of current activation, corresponding to an increase in the spermine-to-spermidine ratio over time. These results provide direct evidence for a role of each polyamine in induction of rectification, and they further demonstrate that in vivo modulation of rectification is possible by manipulation of polyamine levels using genetic and pharmacological approaches.

The purpose of this study was to determine whether luminal polyamines stimulate intestinal mucosal growth in vivo. Rats received 2% alpha-difluoromethylornithine (DFMO) added to their drinking water throughout the experiment. The polyamines spermidine and spermine (3 mg each/100 g body wt) were given intragastrically in combined doses once at 9:30 A.M. and again at 5:30 P.M. Duodenal and jejunal mucosal ornithine decarboxylase (ODC) activity in the DFMO-treated rats was inhibited significantly for the duration of the study. DFMO also markedly decreased the rate of [3H]thymidine incorporation into DNA of duodenal and jejunal mucosa. The decrease in [3H]thymidine incorporation was significant 4 days and maximal 6 and 8 days after beginning treatment with DFMO. Decreased ODC activity and DNA synthesis were paralleled by decreases in total mucosal DNA, RNA, and protein content. Administration of the polyamines significantly reversed the effects of DFMO except the inhibition of ODC. In fact, there were no significant differences in mucosal growth parameters between the controls (without DFMO) and those treated with DFMO plus polyamines. Oral administration of spermidine and spermine at a dose of 4.5 mg each/100 g body wt for 6 days to rats not treated with DFMO increased the normal rate of mucosal growth in the duodenum and jejunum as well. Polyamine accumulation in IEC-6 cells was measured to determine whether it was altered by DFMO. IEC-6 cells took up [3H]putrescine and [3H]spermidine from their surrounding environment and the uptake was stimulated by serum. DFMO (5 mM) totally inhibited the increase in ODC activity but had no effect on the cellular uptake of polyamines in the presence of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is dramatically increased in proliferating cells. In addition to transcriptional regulation of ODC, the present study shows that the enzyme is regulated at the translational level by putrescine and spermidine. ODC synthesis is inhibited by an increase and stimulated by a decrease in their cellular content. Spermidine is a more potent negative regulator than is putrescine. The effects of polyamines on ODC synthesis were not attributable to changes in the cellular content of ODC mRNA, thus demonstrating regulation at the translational level.