Differential topochemistry of three cationic amino acid transporter proteins, hCAT1, hCAT2 and hCAT3, in the adult human brain

The cellular uptake of l-arginine and other cationic amino acids (such as l-lysine and l-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.     

Effect of polyamines and L-ornithine on the development of proembryogenic masses of Cryptomeria japonica

We examined the effects of polyamines, namely, putrescine, spermidine and spermine, and of amino acids, such as l-arginine and l-ornithine, as part of our efforts to identify factors that stimulate the development of proembryogenic masses (PEMs) of Cryptomeria japonica. We maintained two distinct types of PEM designated PEMs A, which consisted of normal embryogenic cells as single embryos with elongated suspensor cells, and PEMs B, which consisted of abnormal embryogenic cells with coalesced embryos on modified Campbell and Durzan medium (mCD) supplemented with individual polyamines at 0–100 μM or amino acids at 0–16.4 mM. All additives had a stimulatory/suppressive effect. Microscopy and image-processing techniques revealed that the regions of authentic embryos of PEMs that were treated with l-ornithine were remarkably enlarged and that the suspensor cells had elongated in the same direction. When all PEMs A were transferred to maturation medium (mCD that contained abscisic acid and maltose at various concentrations), only PEMs that had been treated with l-ornithine matured into somatic embryos and were able to germinate on hormone-free mCD. Our results indicate that l-ornithine is an important stimulator of the development of PEMs to the pre-filamentous stage in C. japonica.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

Direct production of l-ornithine from casein by commercial digestive enzymes and in situ activated arginase

There is a great demand for l-ornithine, which is used as a dietary supplement, and in the pharmaceutical industry. In the present study, when milk casein was hydrolyzed at 37 °C by using commercial digestive enzymes, namely, Pancreatin F and Protease A, a significant accumulation of l-ornithine in the hydrolysate and the simultaneous disappearance of l-arginine was noted. In a radiometric assay, transient but distinct arginase activity, which was sufficiently high for l-ornithine production, was detected in the hydrolysate for a certain period during casein hydrolysis. On the basis of the results of the enzymatic analyses, arginase was thought to be proteolytically generated from an inactive precursor, which may generally be contained in Pancreatin F, and ultimately degraded by further proteolysis. This conversion process using the above-mentioned digestive enzymes is useful for the production of l-ornithine directly from protein sources that are abundant in nature.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Biotechnological production of polyamines by bacteria: recent achievements and future perspectives

In Bacteria, the pathways of polyamine biosynthesis start with the amino acids l-lysine, l-ornithine, l-arginine, or l-aspartic acid. Some of these polyamines are of special interest due to their use in the production of engineering plastics (e.g., polyamides) or as curing agents in polymer applications. At present, the polyamines for industrial use are mainly synthesized on chemical routes. However, since a commercial market for polyamines as well as an industry for the fermentative production of amino acid exist, and since bacterial strains overproducing the polyamine precursors l-lysine, l-ornithine, and l-arginine are known, it was envisioned to engineer these amino acid-producing strains for polyamine production. Only recently, researchers have investigated the potential of amino acid-producing strains of Corynebacterium glutamicum and Escherichia coli for polyamine production. This mini-review illustrates the current knowledge of polyamine metabolism in Bacteria, including anabolism, catabolism, uptake, and excretion. The recent advances in engineering the industrial model bacteria C. glutamicum and E. coli for efficient production of the most promising polyamines, putrescine (1,4-diaminobutane), and cadaverine (1,5-diaminopentane), are discussed in more detail.     

Ornithine dissimilation byTreponema denticola

Treponema denticola convertedl-ornithine, a product ofl-arginine catabolism, to putrescine via a decarboxylation reaction and to proline via a deamination reaction. Ornithine decarboxylation byT. denticola extracts was stimulated by pyridoxal 5′-phosphate. In the absence of pyridoxal 5′-phosphate, (NH₄)₂SO₄-fractionated extracts converted ornithine to proline and ammonia. This activity was not stimulated by α-keto acids, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide or ADP. Neither ornithine δ-transaminase (l-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) nor Δ¹ reductase [l-proline: NAD(P) 5-oxidoreductase, EC 1.5.1.2.] activity was detectable in cell extracts. These results indicate that formation of proline from ornithine inT. denticola is catalyzed by an enzyme system analogous to the ornithine cyclase (deaminating) ofClostridium sporogenes. Exogenous ornithine inhibited the growth ofT. denticola. Thus, in addition to generating putrescine and proline, the ornithine dissimilatory pathways may serve to prevent accumulation of inhibitory concentrations of ornithine in the spirochete's environment.     

Metabolic interactions between restraint stress and <Emphasis Type="SmallCaps">l</Emphasis>-lysine: The effect on urea cycle components

Summary.  We studied the effects of l-lysine on wrap-restraint stress-induced changes in ureagenesis. An exposure to wrap-restraint stress did not affect the plasma concentration of l-lysine, but did decrease plasma urea and arginine. Oral l-lysine (1 g/kg) blocked the effect of stress on ureagenesis, and enhanced the effect of stress on l-arginine. No influence of l-lysine were found in controls. The results imply a stress-specific, ureagenesis-stimulating effect of l-lysine, and suggest an increased requirement for l-arginine during the above conditions. Received December 9, 2002 Accepted January 21, 2003 Published online April 3, 2003 Acknowledgement Authors thank Dr. M. Miura (Ajinomoto Co.) for his help with amino acid analysis and Dr. T. Kimura (Ajinomoto Co.) for discussions on amino acid metabolism. Authors' address: Dr. M. Smriga, Ajinomoto Co. Inc., Institute of Life Sciences, 1-1 Suzuki-cho, 210-8681 Kawasaki, Japan, Fax +81-44-210-5893, E-mail: miroslav_smriga@ajinomoto.com Abbreviations: Arg, l-arginine; Orn, l-ornithine; Lys, l-lysine; p.o., oral; WRS, wrap-restraint stress     

Effects of tensile stress on the α1 nicotinic acetylcholine receptor expression in maxillofacial skeletal myocytes

The present study was to test the hypothesis that 11,12-epoxyeicosatrienoic acid (11,12-EET), a metabolic product of arachidonic acid by cytochrome P450 epoxygenase, regulates nitric oxide (NO) generation of the l-arginine/NO synthase (NOS) pathway in human platelets. Human platelets were incubated in the presence or absence of different concentrations of 11,12-EET for 2 h at 37°C, followed by measurements of activities of the l-arginine/NOS pathway. Incubation with 11,12-EET increased the platelet NOS activity, nitrite production, cGMP content, and the platelet uptake of l-[³H]arginine in a concentration-dependent manner. In addition, 11,12-EET attenuated intracellular free Ca²⁺ accumulation stimulated by collagen, which was at least partly mediated by EET-activated l-arginine/NOS pathway. It is suggested that 11,12-EET regulates platelet function through up-regulating the activity of the l-arginine/NOS/NO pathway.     

Nitric oxide synthesis is blocked in the enteral nervous system during dormant periods of the snail<Emphasis Type="Italic"> Helix lucorum</Emphasis>

During dormancy of terrestrial snails, the whole neuromodulation of the nervous system is deeply modified. In this work we studied the adaptation of a previously described, putatively nitric oxide (NO) forming enteral network to the long-term resting periods of the snail Helix lucorum. The standard NADPH diaphorase (NADPHd) technique, which is an accepted method for histochemical NO synthase (NOS) detection, labeled the same enteric neurons of the midintestine in active or hibernated snails. Quantification of the NO-derived nitrite by the Griess reaction established that the nitrite formation is confined to the NADPHd-reactive network containing the midintestinal segment. In active snails, the nitrite formation could be enhanced by the NOS substrate l-arginine (10 M–1 mM), but decreased by the known NOS inhibitors 1 mM N-nitro-l-arginine (NOARG) and 10 mM aminoguanidine (AG). Application of 1 mM l-arginine and 1 mM NOARG decreased the amplitude of the midintestinal muscle contractile activity, but did not affect the rectal motility. In dormancy, the nitrite formation was reduced in the NADPHd-reactive midintestinal network. Application of l-arginine could not provoke nitrite production and did not influence the midintestinal motility. Our findings indicate that NO is involved in the neural transmission to intestinal muscles of gastropods, but enteric release of NO is blocked during dormancy. The decreased NO synthesis is possibly due to an as yet undefined mechanism, by which the l-arginine/NO conversion ability of NOS could temporarily be inhibited in the long-term resting period of H. lucorum.T. Röszer and Zs. Czimmerer contributed equally to this work as lead authors. This research was supported by OTKA grant no. T42762 (G.B.) and PRCH Student Science Foundation grants 2000, 2002 (T.R.) and 2003 (Zs. Cz). The study is dedicated to Borbála Vecsei-Czimmerer, Elemér Czimmerer, Ágnes M. Fodor-Röszer and József S. Röszer.     

Probing of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine as an Additive for the Temperature-Induced Aggregation of Veterinary Growth Hormones: Fluorescence Study

The influence of l-arginine on the temperature-induced aggregation of porcine and mink growth hormones was studied by fluorescence spectroscopy. It was found that l-arginine suppresses the heat-induced aggregation. Moreover, the analysis of l-arginine interaction with the native proteins by fluorescence spectroscopy and circular dichroism spectroscopy revealed no significant changes in their native structure. On the basis of the results, l-arginine could be considered as a potential additive for the prevention of storage and temperature-related denaturation and aggregation of veterinary growth hormones.     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

Lysinuric protein intolerance mutation is not expressed in the plasma membrane of erythrocytes

Summary We measured mediated fluxes of l-lysine and l-ornithine across the plasma membrane of erythrocytes from control subjects and patients homozygous for the lysinuric protein intolerance (LPI) mutation. We found no differences in net uptake or efflux of cationic amino acids in control and LPI cells, contrary to our findings in cultured skin fribroblasts. We conclude that expression of the LPI (y⁺) transport system for cationic amino acids varies between tissues and that measurements of fluxes in erythrocytes cannot be used for diagnosis of LPI.     

Inhibitory action of serine on growth of bacteria of the genusBacillus on mineral synthetic media

l-Serine added to minimal synthetic media stops the growth ofBacillus subtilis, B. megaterium, B. mycoides andB. pantothenticus. All tested subspecies ofBacillus thuringiensis appear resistant to this amino acid. Serine acts bacteriostatically onB. subtilis strain B 003 and this effect depends on the concentrations of both the amino acid and the plated bacterium. Growth of serine-sensitiveBacillus strains can be restored by simultaneous addition of some other amino acids (e.g. l-threonine,l-arginine,l-aspartate orl-alanine) to the minimal media. This alleviating effect depends on the kind of amino acid. Some amino acids (e.g. l-threonine,l-tyrosine orl-tryptophan) are only effective when the serine concentration is not higher than 125 μmol/L, others (l-arginine,l-proline,l-alanine,l-aspartate orl-glutamate) are effective even when the serine concentration is as high as 500 μmol/L.     

N2-Succinylornithine in ornithine catabolism of Pseudomonas aeruginosa

Most Pseudomonas aeruginosa PAO mutants which were unable to utilize l-arginine as the sole carbon and nitrogen source (aru mutants) under aerobic conditions were also affected in l-ornithine utilization. These aru mutants were impaired in one or several enzymes involved in the conversion of N²-succinylornithine to glutamate and succinate, indicating that the latter steps of the arginine succinyltransferase pathway can be used for ornithine catabolism. Addition of aminooxyacetate, an inhibitor of the N²-succinylornithine 5-aminotransferase, to resting cells of P. aeruginosa in ornithine medium led to the accumulation of N²-succinylornithine. In crude extracts of P. aeruginosa an ornithine succinyltransferase (l-ornithine:succinyl-CoA N²-succinyltransferase) activity could be detected. An aru mutant having reduced arginine succinyltransferase activity also had correspondingly low levels of ornithine succinyltransferase. Thus, in P. aeruginosa, these two activities might be due to the same enzyme, which initiates aerobic arginine and ornithine catabolism.Abbreviations OAT ornithine 5-aminotransferase - SOAT N²-succinylornithine 5-aminotransferase - Oru ornithine utilization - Aru arginine utilization     

Production of Bacillus sphaericus larvicide on industrial peptones

Summary A range of industrial peptones in combination with other carbon and nitrogen sources were investigated in order to define a medium for production of insecticide from Bacillus sphaericus, strain 2362. Industrial protein hydrolysates with an average peptide chain length above 30 were found to be poor substrates. Fermentation of B. sphaericus on protein-based media supplemented with glycerol resulted in production of 4–5x10³ units of larvicide per ml fermentation broth, and toxicity of the larvicide up to 10⁴ units per mg. Equally high larvicide production was obtained, when peptone was substituted with 30 mM l-glutamate and 2 mM proline. Addition of 2 mM l-arginine to this medium prevented sporulation and larvicide production.     

Biotransformation of l-ornithine from l-arginine using whole-cell recombinant arginase

A recombinant arginase was generated for a whole-cell biotransformation system to convert l-arginine to l-ornithine in Escherichia coli. The gene ARG1 coding arginase from Bos taurus liver was synthesized and expressed in E. coli BL21 (DE3) via pETDuet-1. The recombinant arginase was used to catalyze l-arginine to l-ornithine and urea. The reaction was optimal at pH 9.5 and 37 °C. Manganese (10^?5 M) and Emulsifier OP-10 [0.033 % (v/v)] could promote arginase activity. In a scale up study, l-arginine conversion rate reached 98 % with a final concentration of 111.52 g l-ornithine/l.     

Catalysis on dinuclear Mn(II) centers: hydrolytic and redox activities of rat liver arginase

 Rat liver arginase contains a dinuclear Mn₂(II,II) center in each subunit having EPR properties similar to those observed in Mn-catalases. The principal physiologic role of arginase is catalyzing the hydrolytic cleavage of l-arginine to produce l-ornithine and urea. Here we demonstrate that arginase catalyzes the disproportionation of hydrogen peroxide by a redox mechanism analogous to Mn-catalases, but at rates that are 10^–5 to 10^–6 of k _cat for the Mn-catalases, and also exhibits peroxidase activity. The dinuclear Mn₂(II,II) center is essential for maximal catalase activity, since both the H101N and H126N mutant arginases containing only one Mn(II)/subunit have catalase activities that are <3% of that for the wild-type enzyme. Like the Mn-catalases, the catalase activity of arginase is not inhibited by millimolar concentrations of CN^–, the most potent inhibitor of heme catalases, or by EDTA, a chelator of free metal ions. The catalase activity of arginase is not significantly inhibited by Cl^– or F^–, in contrast to Mn-catalases, while potent inhibitors of the hydrolytic activity are also effective inhibitors of the catalase activity. These results suggest that lower affinity of hydrogen peroxide to the active site of arginase contributes to the lower catalase activity. EPR spectroscopy reveals that potent inhibitors of the hydrolytic reaction, including N ^ω-hydroxy-l-arginine, l-lysine, and l-valine, decouple the electronic interaction between the Mn²⁺ ions, most probably by removing a μ-bridging ligand or by increasing the intermanganese separation. The capacity for arginase to deliver a hydroxide ion to hydrolyze the l-arginine substrate is suggested to arise from a "dinuclear effect", wherein the two metal ions contribute more or less equivalently in deprotonation of metal-bound water molecule. Structure-reactivity analyses of these reactions will provide insights into the factors that control redox versus hydrolytic function in dimanganese clusters. Received: 18 November 1996 / Accepted: 7 April 1997     

Inhibitors of N α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency

A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N ^α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC₅₀ values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC₅₀ value of 85 μM while N ^α-trifluoroacetyl-l-ornithine (1f), N ^α-ethoxycarbonyl-l-ornithine (2b), and N ^α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC₅₀ values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N ^α-acetyl-d-ornithine (1a) and N ^α-fluoro- (1f), N ^α-chloro- (1g), N ^α-dichloro- (1h), and N ^α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC₅₀ values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.     

Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.