The transport of putrescine and spermidine into Leishmnania donovani promastigotes and Leishmania mexicana promastigotes and amastigotes has been characterised. Polyamine transport was shown to be saturable and temperature-sensitive for both developmental stages of Leishmania. Transport was pH-dependent with pH optima of 7.4 and 5.5 for promastigotes and amastigotes, respectively. The uptake process was independent of extracellular Na+, but inhibited by protonophores and H+-ATPase inhibitors. Kinetic analyses of polyamine transport showed that Km and Vmax differed between promastigotes of the two species and between promastigotes and amastigotes of L. mexicana. Inhibition data suggest that putrescine and spermidine use different transporters. The aromatic diamidine pentamidine, the drug of choice for treatment of antimonial-resistant cases of leishmaniasis, inhibited both putrescine and spermidine transport non-competitively.

2-Difluoromethylornithine (DFMO) was administered to 1,2-dimethylhydrazine (DMH)-treated mice to reduce colonic polyamine levels and mucosal hyperplasia. Mice received 1% DFMO in drinking water throughout the experiment and were given injections of DMH (20 mg/kg) weekly for 28 weeks. DFMO inactivated 93% of colonic ornithine decarboxylase activity. Although DMH treatment did not induce colonic ornithine decarboxylase activity by Week 28, the putrescine content was increased 31% in DMH-treated mice (p less than 0.01). Concurrent treatment with DFMO depressed putrescine content (42 to 63%) and spermidine content (27 to 38%), but it increased spermine content (18 to 22%). At Week 28 of treatment with DMH alone, RNA content was increased 8.6% (p less than 0.01), DNA content 10% (p less than 0.01), DNA specific activity 24% (p less than 0.01), and crypt depth 20% (p less than 0.01), but not in mice receiving DMH and DFMO. At 28 weeks, 13 of 17 mice (76%) treated with DMH alone had histologically confirmed colon cancers; of mice treated with DMH and DFMO, two of 18 (11%) had colonic tumors. Throughout the experiment, 50 colon cancers developed in 16 DMH-treated mice (mean, 3.12 tumors/mouse); three mice treated with DMH and DFMO developed three colon cancers total (p less than 0.001). Reduction of colonic polyamine levels after DFMO treatment prevents proliferative changes induced by DMH and reduces the incidence of tumors.

The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H2O2 and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H2O2 generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.

The naturally occurring polyamines, spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2] and spermidine [NH2(CH2)3NH(CH2)4NH2], as well as the diamine putrescine [NH2(CH2)4NH2], are widely spread in nature. They occur in plants, micro-organisms and animal tissues and fulfil many important physiological functions. Due to their cationic nature they interact with negatively charged macromolecules such nucleic acids, phospholipids and proteins. This ionic interaction, which is reversible, leads to the stabilization of DNA, tRNA, membranes and some proteins. Early studies demonstrated that polyamines stimulate the growth of pro- and eukaryotic cells and that they play an important role in carcinogenesis and in malignant transformation processes. As a result of these studies various inhibitors of polyamine biosynthesis have been synthesized and are used to combat cancer and parasitic diseases (e.g., African sleeping sickness).

OBJECTIVE

Combination of polyamine and prostaglandin E2 (PGE2)-synthesis inhibitors reduced the risk of colorectal adenoma (CRA) by 70% in patients who received polypectomies. We studied effects of the combination of difluoromethylornithine (DFMO) and sulindac on biomarkers and investigated factors that modify their efficacy.

METHODS

We analyzed rectal mucosal levels of polyamines (spermidine, spermine, and putrescine) and PGE2, treatment regimens, and risk of CRA in 267 participants of a phase IIb/III chemoprevention trial of DFMO/sulindac.

RESULTS

In the group that received DFMO/sulindac, spermidine-to-spermine ratio (Spd:Spm) in rectal mucosa decreased between baseline and 12- and 36-month follow-up examinations (0.30, 0.23, and 0.24, respectively; P < .001 for both comparisons to baseline). Putrescine levels decreased between baseline and 12 months (0.46 vs 0.15 nmol/mg protein; P < .001) but rebounded between 12 and 36 months (0.15 vs 0.36 nmol/mg protein; P = .001). PGE2 levels did not change, although aspirin use was significantly associated with lower baseline levels of PGE2. No significant associations were observed between changes in biomarker levels and efficacy. However, drug efficacy was greatest in subjects with low Spd:Spm and high PGE2 at baseline; none of these subjects, versus 39% of those given placebo, developed CRA (P < .001). Efficacy was lowest in subjects with high Spd:Spm and low PGE2 at baseline; 28% developed CRA, compared with 36% of patients given placebo (P = .563).

CONCLUSIONS

A combination of DFMO and sulindac significantly suppressed production of rectal mucosal polyamines but not PGE2. No relationship was found between changes in biomarker levels and response. However, baseline biomarker levels modified the effect of DFMO/sulindac for CRA prevention.

BACKGROUND

Enormous work has shown that polyamines are involved in a variety of physiological processes, but information is scarce on the potential of modifying disease response through genetic transformation of a polyamine biosynthetic gene.

RESULTS

In the present work, an apple spermidine synthase gene (MdSPDS1) was introduced into sweet orange (Citrus sinensis Osbeck 'Anliucheng') via Agrobacterium-mediated transformation of embryogenic calluses. Two transgenic lines (TG4 and TG9) varied in the transgene expression and cellular endogenous polyamine contents. Pinprick inoculation demonstrated that the transgenic lines were less susceptible to Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker, than the wild type plants (WT). In addition, our data showed that upon Xac attack TG9 had significantly higher free spermine (Spm) and polyamine oxidase (PAO) activity when compared with the WT, concurrent with an apparent hypersensitive response and the accumulation of more H₂O₂. Pretreatment of TG9 leaves with guazatine acetate, an inhibitor of PAO, repressed PAO activity and reduced H₂O₂ accumulation, leading to more conspicuous disease symptoms than the controls when both were challenged with Xac. Moreover, mRNA levels of most of the defense-related genes involved in synthesis of pathogenesis-related protein and jasmonic acid were upregulated in TG9 than in the WT regardless of Xac infection.

CONCLUSIONS

Our results demonstrated that overexpression of the MdSPDS1 gene prominently lowered the sensitivity of the transgenic plants to canker. This may be, at least partially, correlated with the generation of more H₂O₂ due to increased production of polyamines and enhanced PAO-mediated catabolism, triggering hypersensitive response or activation of defense-related genes.

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) alpha-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) alpha-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibitors require at least two amine groups to be effective. For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups. Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction. From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore. Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine. Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction.

Ornithine decarboxylase (ODC) catalyses the first step in the synthesis of the polyamines putrescine, spermidine and spermine. The polyamines are essential for cell growth, but at elevated levels they may be tumorigenic, toxic, or may induce apoptosis. Therefore, ODC activity is highly regulated. It is induced when cells are stimulated to grow, and it is subjected to feedback inhibition by the polyamines. By causing ribosomal frameshifting, polyamines induce the synthesis of antizyme, a 23-kDa protein, which binds to ODC, inhibits its activity and promotes its degradation by the 26 S proteasome. Antizyme, in turn, is inhibited by antizyme inhibitor (AZI). We describe the cloning of a mouse AZI cDNA, encoding a protein with high homology to mouse ODC. Using purified recombinant proteins, we show that AZI (which has no ODC activity) can release enzymically active ODC from antizyme suppression in vitro. We also show that ODC reactivation takes place in mouse fibroblasts upon transient transfection with an AZI-expressing plasmid construct. Finally we demonstrate that the AZI mRNA content of mouse fibroblasts increases significantly within an hour of growth stimulation, i.e. much earlier than ODC transcripts. Our results indicate that induction of AZI synthesis may represent a means of rescuing ODC molecules that have been inactivated and tagged for degradation by antizyme, when culture conditions improve and polyamine production is needed for cell growth and proliferation.

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.

Bacterial growth was measurably slowed by a combination of drugs which inhibit polyamine-biosynthetic enzymes. Addition of DL-alpha-monofluoromethylornithine, which was shown to inactivate irreversibly ornithine decarboxylase extracted from Escherichia coli (Ki = 0.36 mM) and Pseudomonas aeruginosa (Ki = 0.30 mM), DL-alpha-difluoromethylarginine and dicyclohexylammonium sulphate to cultures of E. coli or P. aeruginosa resulted in a 40 and 70% increase in generation times (decreased growth rates) respectively, which was completely reversed by the addition of 0.1 mM-putrescine plus 0.1 mM-spermidine to the medium. Decreased intracellular polyamine concentrations correlated with increased generation times; putrescine concentration was decreased by 70% in E. coli and 80% in P. aeruginosa, while spermidine concentration was decreased by 50% in E. coli and 95% in P. aeruginosa. Subsequent investigation of the inactivation of the ornithine decarboxylase by monofluoromethylornithine indicated that it was active-site directed, as the normal substrate ornithine slowed the rate of inhibition. Specific interference with polyamine biosynthesis may be a viable approach to control of some bacterial infections.

Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of approximately 1.7 or approximately 2 for DNA that had been gamma-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

Spermidine/spermine N1-acetyltransferase (SSAT), the rate-controlling enzyme in the interconversion of spermidine and spermine, is regulated by polyamines and their analogs at many levels of gene expression. Recently, SSAT pre-mRNA has been shown to undergo alternative splicing by inclusion of an exon that contains premature termination codons. In the present study, we show that alterations in the intracellular polyamine level resulted in a change in the relative abundance of SSAT transcripts. Addition of polyamines or their N-diethylated analogs reduced the amount of the variant transcript, whereas polyamine depletion by 2-difluoromethylornithine or MG-132 enhanced the exon inclusion. Experiments performed with protein synthesis inhibitors and siRNA-mediated down-regulation of Upf1 protein verified that the variant transcript was degraded by nonsense-mediated mRNA decay (NMD). Interestingly, several proteins have been shown to regulate their expression by alternative splicing-coupled NMD, termed regulated unproductive splicing and translation (RUST). Our present results suggest that in the case of SSAT, RUST is mediated by polyamines, and this system functions to fine-tune the polyamine metabolism.

Triplex DNA has been recently studied as an anti-gene strategy to suppress the transcription of specific genes. A major challenge in this area is to stabilize triplex DNA at physiological conditions. We studied the effects of putrescine, spermidine, spermine, their synthetic homologs, and their acetyl derivatives on triple- and double-stranded structures formed from poly(dA) and poly(dT) by measuring their respective melting temperatures. In the presence of polyamines, the absorbance (A) versus temperature (T) profile showed two transitions: Tm1, corresponding to triplex->>duplex+single-stranded DNA, and Tm2, corresponding to duplex melting. In the presence of 0.5 mM putrescine, Tm1 and Tm2 were 44.8 and 71 degrees C, respectively, in 10 mM sodium cacodylate buffer (pH 7.2). In contrast, triplex DNA was not detectable when the A versus T profile of the polynucleotides was monitored in the absence of putrescine. Tm2 was also lower (55 degrees C) in the absence of putrescine. With 2.5 microM spermidine or 0.1 microM spermine, Tm1 values were 42.8 and 54.4 degrees C and Tm2 values were 65 and 82 degrees C, respectively. As the concentration of polyamine was increased, the difference between Tm1 and Tm2 decreased, and both melting transitions merged into one Tm, corresponding to the melting of triplex DNA to single strands. In a series of putrescine homologs, H2N (CH2)nNH2 where n = 2-6 (n = 4 for putrescine), H2N(CH2)3NH2 was the most effective diamine to stabilize the poly(dA).2poly(dT) triplex. At 10 mM concentration, diaminopropane stabilized the triplex DNA such that the Tm1 was 10 degrees C higher than that in the presence of an equimolar concentration of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the self-cleavage reaction performed by a ribozyme that contains 164 nucleotides of Neurospora VS RNA. Self-cleavage requires a divalent cation, magnesium being more effective than manganese or calcium. Spermidine or monovalent cations stimulate the reaction but cannot replace magnesium. The temperature optimum is rather broad, around 40-50 degrees C. Unlike some other ribozymes, VS self-cleavage is inhibited by even low concentrations of urea or formamide. The rate of cleavage is the same from pH 5.5 to 8.9, suggesting either that hydroxide is not directly involved in the cleavage chemistry or that a step that precedes the actual cleavage event is rate-limiting.

A strain of Escherichia coli was constructed in which all of the genes involved in polyamine biosynthesis--speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase)--had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.

The genome sequence of the hyperthermophilic methanogen Methanococcus jannaschii contains homologs of most genes required for spermidine polyamine biosynthesis. Yet genomes from neither this organism nor any other euryarchaeon have orthologs of the pyridoxal 5'-phosphate-dependent ornithine or arginine decarboxylase genes, required to produce putrescine. Instead, as shown here, these organisms have a new class of arginine decarboxylase (PvlArgDC) formed by the self-cleavage of a proenzyme into a 5-kDa subunit and a 12-kDa subunit that contains a reactive pyruvoyl group. Although this extremely thermostable enzyme has no significant sequence similarity to previously characterized proteins, conserved active site residues are similar to those of the pyruvoyl-dependent histidine decarboxylase enzyme, and its subunits form a similar (alphabeta)(3) complex. Homologs of PvlArgDC are found in several bacterial genomes, including those of Chlamydia spp., which have no agmatine ureohydrolase enzyme to convert agmatine (decarboxylated arginine) into putrescine. In these intracellular pathogens, PvlArgDC may function analogously to pyruvoyl-dependent histidine decarboxylase; the cells are proposed to import arginine and export agmatine, increasing the pH and affecting the host cell's metabolism. Phylogenetic analysis of Pvl- ArgDC proteins suggests that this gene has been recruited from the euryarchaeal polyamine biosynthetic pathway to function as a degradative enzyme in bacteria.

Until recently, a capacity for apoptosis and synthesis of nitric oxide *NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated L-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with L-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite *NO production. These effects were inhibited by the *NO synthase inhibitor N(omega)-nitroarginine methyl ester (L-NAME). Exogenous *NO limited DNA fragmentation but did not restore proliferation rates. Because L-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited L-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by L-arginine by means of (i) a *NO synthase-dependent *NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

Leakage of ions (Na+, K+) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by 'pore-forming' agent has been studied. Leakage from intact cells is induced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells--or across phospholipid bilayers modified by agent--is prevented by low concentrations of the same cations and by higher concentrations of Ca2+, Mn2+ or Ba2+; Mg2+, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca2+) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na+, K+, Rb+, NH4+) and divalent (Mg2+, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn2+ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.