The exchange of ribosomal subunits during the release of growing polypeptide chains by puromycin has been investigated in a bacterial cell-free system engaged in protein synthesis. The addition of spermidine, used as a stabilizing agent of 70S monomers, caused a strong inhibition of the subunit exchange. This result led us to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. This behavior is different from that occurring during physiological termination of translation, where the ribosomes detach in a dissociated form. Some implications of the postulated mechanism are also discussed.

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.

Recent investigations on plant polyamine oxidase (PAO) are reviewed. The enzyme belongs to a new class of flavoenzymes with similar structural features including, among others, monoamine oxidase. Plant PAOs catalyse the oxidation of the polyamine substrates spermidine and spermine. The reaction products are propane-1,3-diamine and 1-pyrroline or 1-(3-aminopropyl)pyrrolinium, respectively, along with hydrogen peroxide. Plant PAOs are predominantly localised in the cell wall. Purification procedures and molecular properties of several plant PAOs are compared. A special attention is being paid to the recently solved crystal structure of the maize enzyme and its implications for the substrate binding and catalytic mechanism. Substrate specificity and inhibitors of plant PAOs are also described. The potential roles for PAO-generated H(2)O(2) in lignin biosynthesis and cell wall cross-linking reactions, which may regulate growth and contribute to cell defence, are discussed.

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.

Furfural and acetic acid are two prevalent inhibitors to microorganisms during cellulosic ethanol production, but molecular mechanisms of tolerance to these inhibitors are still unclear. In this study, genome-wide transcriptional responses to furfural and acetic acid were investigated in Saccharomyces cerevisiae using microarray analysis. We found that 103 and 227 genes were differentially expressed in the response to furfural and acetic acid, respectively. Furfural downregulated genes related to transcriptional control and translational control, while it upregulated stress-responsive genes. Furthermore, furfural also interrupted the transcription of genes involved in metabolism of essential chemicals, such as etrahydrofolate, spermidine, spermine, and riboflavin monophosphate. Acetic acid downregulated genes encoding mitochondrial ribosomal proteins and genes involved in carbohydrate metabolism and regulation and upregulated genes related to amino acid metabolism. The results revealed that furfural and acetic acid had effects on multiple aspects of cellular metabolism on the transcriptional level and that mitochondria might play important roles in response to both furfural and acetic acid. This research has provided insights into molecular response to furfural and acetic acid in S. cerevisiae, and it will be helpful to construct more resistant strains for cellulosic ethanol production.

Using Northern blotting, the expression levels of the genes for polyamine metabolism regulatory proteins and clusterin have been measured in a series of 23 human prostate cancers (CaPs) dissected from radical prostatectomy specimens. Patient matched, nontumor tissue was dissected from benign areas of the gland. The results indicate that transcripts encoding ornithine decarboxylase (ODC), ODC antizyme, adenosylmethionine decarboxylase, and spermidine/spermine N1-acetyltransferase (SSAT) were significantly higher, whereas clusterin (sulfated glycoprotein 2) mRNA was significantly lower in tumors compared with the benign tissue. All mRNA levels were compared with those of histone H3 and growth arrest-specific gene 1, markers of cell proliferation and cell quiescence, respectively, and glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene. In poorly differentiated and locally invasive CaPs and in tumors with unfavorable prognosis or total prostate-specific antigen (PSA) levels>> 10.0 ng/ml at diagnosis, an overall increase in the levels of H3 mRNA and a decrease in growth arrest-specific gene 1 mRNA was detected, indicative of higher proliferation activity, whereas the differences in expression levels for the polyamine metabolism and clusterin genes were higher. ODC and SSAT changes were positively correlated in normal tissue but not in high-grade cancer, whereas ODC antizyme and SSAT changes were positively correlated in more malignant CaPs but not in normal tissue. Tumor classification based on the changes in expression levels of all of the genes studied could be correlated to differentiation grade and local invasiveness classification systems in 72.2 and 83.3% of the cases, respectively. In a 1-year follow-up period, three patients whose CaPs ranked as less aggressive according to clinical staging, but classified as advanced cancers with the proposed molecular classification, showed increases in total PSA levels, indicative of tumor relapse. Thus, molecular classification, based on gene expression, may enhance the available prognostic tools for prostate tumors.

A denitrifying bacterium, designated strain FS(T), was isolated from anoxic digested sludge on oestradiol [17beta-oestra-1,3,5(10)-triene-3,17-diol] or testosterone (17beta-hydroxyandrost-4-en-3-one) as the sole source of carbon and energy with nitrate as the electron acceptor. Strain FS(T) represents the first known bacterium to grow anaerobically on both oestradiol (C-18) and testosterone (C-19). Steroidal hormones were degraded completely by nitrate reduction to dinitrogen monoxide, which was further reduced to dinitrogen in stationary-phase cultures. Gram-negative cells were slightly curved rods, 0.3-0.5 x 0.6-1.6 microm in size, motile, non-fermentative, non-spore-forming and catalase- and oxidase-positive, showing optimal growth at pH 7.0, 28 degrees C and 0.1% (w/v) NaCl. Beside steroidal hormones, the bacterium utilized only a narrow range of organic substrates with nitrate as the electron acceptor, including several fatty acids and glutamate. No aerobic or anaerobic growth occurred on liquid or solid complex media. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain FS(T) has no known close relatives and represents a distinct lineage within the Gammaproteobacteria. Together with the genera Nevskia, Hydrocarboniphaga, Solimonas and Sinobacter (less than 88% 16S rRNA gene sequence similarity to strain FS(T)), it forms a phylogenetic cluster separated from the families Chromatiaceae, Ectothiorhodospiraceae and Xanthomonadaceae. The quinone system of strain FS(T) consisted exclusively of ubiquinone Q-8. The dominant polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Spermidine in combination with putrescine and traces of sym-homospermidine were the basic polyamines. The major fatty acids detected in testosterone- or heptanoate-grown cells were C(15:0) and C(17:1)omega8c, minor hydroxylated fatty acids were C(11:0) 3-OH and C(12:0) 3-OH. The G+C content of the DNA was 61.9 mol%. Based on the high 16S rRNA gene sequence divergence and different phenotypic properties from previously described gammaproteobacteria in combination with chemotaxonomic data, strain FS(T) is considered to represent a new genus and species, for which the name Steroidobacter denitrificans gen. nov., sp. nov. is proposed. The type strain of Steroidobacter denitrificans is FS(T) (=DSM 18526(T) =JCM 14622(T)).

Aminopropyltransferases transfer aminopropyl groups from decarboxylated S-adenosylmethionine to amine acceptors, forming polyamines. Structural and biochemical studies have been carried out with the human spermidine synthase, which is highly specific for putrescine as the amine acceptor, and the Thermotoga maritima spermidine synthase, which prefers putrescine but is more tolerant of other substrates. Comparison of the structures of the human spermidine synthase with both substrates and products with the known structure of T. maritima spermidine synthase complexed to a multisubstrate analogue inhibitor and analysis of the properties of site-directed mutants provide a general mechanistic hypothesis for the aminopropyl transfer reaction. The studies also provide a structural basis for the specificity of the spermidine synthase subclass of the aminopropyltransferase family.

Thiol-dependent hydroperoxide metabolism in parasites is reviewed in respect to potential therapeutic strategies. The hydroperoxide metabolism of Crithidia fasciculata has been characterized to comprise a cascade of three enzymes, trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase, plus two supportive enzymes to synthesize the redox mediator trypanothione from glutathione and spermidine. The essentiality of the system in respect to parasite vitality and virulence has been verified by genetic approaches. The system appears to be common to all genera of the Kinetoplastida. The terminal peroxidase of the system belongs to the protein family of peroxiredoxins which is also represented in Entamoeba and a variety of metazoan parasites. Plasmodial hydroperoxide metabolism displays similarities to the mammalian system in comprising glutathione biosynthesis, glutathione reductase, and at least one glutathione peroxidase homolog having the active site selenocysteine replaced by cysteine. Nothing precise is known about the antioxidant defence systems of Giardia, Toxoplasma, and Trichomonas species. Also, the role of ovothiols and mycothiols reportedly present in several parasites remains to be established. Scrutinizing known enzymes of parasitic antioxidant defence for suitability as drug targets leaves only those of the trypanosomatid system as directly or indirectly validated. By generally accepted criteria of target selection and feasibility considerations tryparedoxin and tryparedoxin peroxidase can at present be rated as the most appealing target structures for the development of antiparasitic drugs.

Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5-10-fold decreases in glutathionylspermidine and trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in trypanothione reductase and gamma-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.

Synaptic activity causes reductions in cleft [Ca(2+)] that may impact subsequent synaptic efficacy. Using modified patch-clamp techniques to record from single neocortical nerve terminals, we report that physiologically relevant reductions of extracellular [Ca(2+)] ([Ca(2+)](o)) activate voltage-dependent outward currents. These outward currents are carried by a novel nonselective cation (NSC) channel that is indirectly inhibited by various extracellular agents (rank order potency, Gd(3+)>> spermidine>> Ca(2+)>> Mg(2+), typical for [Ca(2+)](o) receptors). The identification of a Ca(2+) sensor-NSC channel pathway establishes the existence of a mechanism by which presynaptic terminals can detect and respond to reductions in cleft [Ca(2+)]. Activation of NSC channels by falls in [Ca(2+)](o) would be expected during periods of high activity in the neocortex and may modulate the excitability of the presynaptic terminal.

Biological transmethylation reactions and polyamine biosynthesis share the substrate S-adenosyl-L-methionine. Under normal conditions, decarboxylated S-adenosyl-L-methionine, the aminopropyl donor for polyamine biosynthesis, does not accumulate because of its rapid utilization in spermidine and spermine synthesis. Alteration of polyamine synthesis by DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of L-ornithine decarboxylase, leads to a striking accumulation of decarboxylated S-adenosyl-L-methionine in rat hepatoma cells cultured in vitro and in rat ventral prostate. This increase is due both to lack of putrescine and spermidine for the aminopropyltransferase reactions and to the elevation of S-adenosyl-L-methionine decarboxylase activity. The biological implications of accumulation of decarboxylated S-adenosyl-L-methionine are discussed with regard to the regulation of S-adenosyl-L-methionine decarboxylase activity and to the antiproliferative effects of DL-alpha-difluoromethylornithine.

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Poly(arginine), poly(lysine) and poly(ornithine) induce histamine secretion from human basophil leukocytes in the concentration range 1--100 nmol/l. Histamine secretion induced by poly(arginine) requires extracellular calcium at 0.1--1 mmol/l. Strontium (1--10 mmol/l) will substitute for calcium. Lanthanum (30--90 nmol/l) inhibits histamine release induced by poly(arginine). Histamine secretion induced by poly(arginine) is inhibited by 1--30 mumol/l N-ethyl-maleimide, 0.3--3 mmol/l 2-deoxy-D-glucose, 0.3--3 mmol/l dibutyryl cyclic AMP, 0.3--3 mmol/l, adenosine 3'5'-cyclicphosphorothioate. The action of poly(arginine) is inhibited by pretreatment of basophils at 47 degrees C or with neuraminidase. 10 microgram/ml heparin inhibits the response to poly(arginine). Histamine releasing potency of the polymer amino acids is dependent on chain length of the peptide. Succinylated poly(lysine) is inactive. Monomer amino acids do not release histamine and do not inhibit the action of the polymers. Histones and protamine do not release histamine, nor do the peptides eledoisin and tuftsin. Putrescine, cadaverine, spermine and spermidine do not release histamine. Poly(glutamic acid), poly(aspartic acid) and poly(tyrosine) are also inactive. The IgE-mediated release of histamine appears to be independent of that mediated by poly(arginine).

1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.

With the recent discovery of the polyamine catabolic enzyme spermine oxidase (SMO/PAOh1), the apparent complexity of the polyamine metabolic pathway has increased considerably. Alone or in combination with the two other known members of human polyamine catabolism, spermidine/spermine N(1)-acetyltransferase, and N(1)-acetylpolyamine oxidase (PAO), SMO/PAOh1 expression has the potential to alter polyamine homeostasis in response to normal cellular signals, drug treatment and environmental and/or cellular stressors. The activity of the oxidases producing toxic aldehydes and the reactive oxygen species (ROS) H(2)O(2), suggest a mechanism by which these oxidases can be exploited as an antineoplastic drug target. However, inappropriate activation of the pathways may also lead to pathological outcomes, including DNA damage that can lead to cellular transformation. The most recent data suggest that the two polyamine catabolic pathways exhibit distinct properties and understanding these properties should aid in their exploitation for therapeutic and/or chemopreventive strategies.

The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.

BACKGROUND

Polyamines (e.g., putrescine, spermidine, and spermine) are required for optimal cell growth. Inhibition of polyamine synthesis suppresses carcinogen-induced epithelial cancers, including colon cancer, in animal models. In a short-term phase IIa trial, we determined that low doses of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (an enzyme involved in polyamine synthesis), reduced the polyamine content of normal-appearing rectal mucosa of subjects with a prior history of resected colon polyps. In a follow-up study, we have attempted to determine the lowest dose of DFMO that can suppress the polyamine content of rectal mucosa over a course of 1 year with no or minimal side effects.

METHODS

Participants were randomly assigned to daily oral treatment with a placebo or one of three doses (0.075, 0.20, or 0.40 g/m2) of DFMO. Baseline and serial determinations of polyamine levels in rectal mucosa and extensive symptom monitoring (including audiometric measurements, since DFMO causes some reversible hearing loss at higher doses) were performed over a 15-month period.

RESULTS

DFMO treatment reduced putrescine levels in a dose-dependent manner. Following 6 months of treatment, doses of 0.20 and 0.40 g/m2 per day reduced putrescine levels to approximately 34% and 10%, respectively, of those observed in the placebo group. Smaller decreases were seen in spermidine levels and spermidine:spermine ratios. Polyamine levels increased toward baseline values after discontinuation of DFMO. Although there were no statistically significant differences among the dose groups with respect to clinically important shifts in audiometric thresholds and nonaudiologic side effects, statistically significant higher dropout and discontinuation rates were observed in the highest dose group.

CONCLUSIONS

Polyamine levels in rectal mucosa can be continuously suppressed by daily oral doses of DFMO that produce few or no side effects. A dose of 0.20 g/m2 can be used safely in combination phase IIb or single-agent phase III chemoprevention trials.

The natural polyamines (putrescine, spermidine and spermine) are ubiquitous low-molecular aliphatic amines that play multifunctional roles in cell growth and differentiation. Recently, evidence has merging that polyamines are actively involved in cell death. Changes in polyamine homeostasis have been reported during cell death of nerve cells, in programmed cell death of embryonic cells and in various in vitro models of apoptosis. Polyamines and many of their structural analogs exert cytotoxic effects in vitro as well in vivo. Furthermore, polyamine analogs and inhibitors of the polyamine anabolic/catabolic pathways modulate processes of cell death in a cell-type specific way. Much ambiguity exists in the working mechanisms by which polyamines mediate apoptosis since they have been shown to act as promoting, modulating or protective agents in apoptosis. Nevertheless, from the studies reviewed here it can be concluded that polyamines are critically involved in cellular survival which makes them suitable targets for therapeutic intervention that is specifically directed to cell death pathways.

The bis(glutathionyl)spermidine trypanothione exclusively occurs in parasitic protozoa of the order Kinetoplastida, such as trypanosomes and leishmania, some of which are the causative agents of several tropical diseases. The dithiol is kept reduced by the flavoenzyme trypanothione reductase and the trypanothione system replaces in these parasites the nearly ubiquitous glutathione/glutathione reductase couple. Trypanothione is a reductant of thioredoxin and tryparedoxin, small dithiol proteins, which in turn deliver reducing equivalents for the synthesis of deoxyribonucleotides as well as for the detoxification of hydroperoxides by different peroxidases. Depending on the individual organism and the developmental state, the parasites also contain significant amounts of glutathione, mono-glutathionylspermidine and ovothiol, whereby all four low molecular mass thiols are directly (trypanothione and mono-glutathionylspermidine) or indirectly (glutathione and ovothiol) maintained in the reduced state by trypanothione reductase. Thus the trypanothione system is central for any thiol regeneration and trypanothione reductase has been shown to be an essential enzyme in these parasites. The absence of this pathway from the mammalian host and the sensitivity of trypanosomatids toward oxidative stress render the enzymes of the trypanothione metabolism attractive target molecules for the rational development of new drugs against African sleeping sickness, Chagas' disease and the different forms of leishmaniasis.

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.

In our earlier work we showed that either spermidine or spermine could support the growth of spe2Delta or spe3Delta polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3Delta fms1Delta mutant that lacked both spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by spermidine; i.e., spermine was not effective because it cannot be oxidized to spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.

The present study suggests that newly synthesized histamine is involved in the development of some animal tumors (e.g., Lewis lung carcinoma in mice and Morris hepatoma in rats). A marked induction of histidine decarboxylase (HDC) and an increase in the histamine concentration were observed in the tumors approximately 1 week after inoculation, and there were parallel increases in ornithine decarboxylase activity and the concentrations of polyamines. The H2 receptor antagonist, cimetidine, significantly reduced tumor growth in the animal models while the H1 receptor antagonist, dexchlorpheniramine, had no effect, suggesting that histamine could act via H2 receptor sites. Extensive depletion of tumor histamine induced by local injection of Compound 48/80 did not result in a significant cytostatic effect. Monofluoromethylhistidine (MFMH), an enzyme-activated irreversible inhibitor of HDC, retarded the growth of hepatoma tissue culture cells grown in culture, and when infused s.c. at 60 mg/kg/day it greatly inhibited the development of tumors induced i.m. by hepatoma tissue culture cells in Buffalo rats. MFMH also had pronounced antitumoral effects on EMT6 sarcomas and Lewis lung carcinomas in mice, which were associated with inhibition of HDC and depletion of the histamine content of the tumors. These cytostatic effects were clearly enhanced when MFMH was combined in therapy with the specific ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine. The antitumoral effects of the combination were associated with marked decreases in the tumor histamine and putrescine contents. It is proposed that nascent histamine, like newly synthesized putrescine and spermidine, plays a role in the rapid proliferation of animal tumors. Inhibition of HDC by essentially nontoxic drugs such as MFMH could represent a novel approach to the control of neoplastic growth.