The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and ⌬arg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The ⌬arg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the ⌬arg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the ⌬arg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.

a b s t r a c tIn-situ irradiations with 150 keV W þ ions have been performed on W and W-5wt.% (Re; Ta; V) alloys in a comprehensive study of the influences of irradiation temperature T irr , dose, alloying elements and grain orientations on radiation damage production and microstructural evolution. For T irr between 30 K and 1073 K, the first observable defects in pure W appeared at doses 0.01 dpa, and were most likely vacancy loops, with Burgers vectors predominantly of type b ¼ ½ <111>. With increasing T irr , the retained defect concentration decreased strongly and the maximum cluster size increased from~1300 point defects at 30 K to~2300 point defects at 1073 K. At all irradiation temperatures, the evolution of damage microstructures with dose from 0.1 to 1.0 dpa involved defect cluster migration, with mutual elastic interactions often leading to spatial inhomogeneities and loop reactions. In pure W, spatial ordering of loops was observed at doses >0.4 dpa and T irr ! 773 K in grains close to z ¼ <001>. No such ordering was found in similar grain orientations for the W-(Re; Ta) alloys, but it was found in the non-z ¼ <001> grains. Post-irradiation analysis on W and W-5 wt% (Re; Ta) at 1.0 dpa showed that ½ <111> and <100> loops of both vacancy and interstitial type were present, at number densities~10 15 loops m À2 . In all cases ½ <111> loops were dominant, the fraction of these with interstitial nature increased with T irr , and the proportion of <100> loops decreased with increasing T irr . Compared with pure W, microstructures in the W-(Re; Ta) alloys exhibited higher loop number densities and evolved more quickly with increasing dose towards damage saturation.

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of ⌬odc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The ⌬odc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the ⌬odc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.Polyamines are cationic compounds that play essential roles in cell proliferation, differentiation, and macromolecular synthesis (1-3). Ornithine decarboxylase (ODC) 1 catalyzes the conversion of ornithine to putrescine (1,4-diaminobutane) and is the initial and rate-limiting enzyme in polyamine biosynthesis in most organisms (4). The ODC enzyme of protozoan parasites is a novel therapeutic target, because D,L-␣-difluoromethylornithine (DFMO; eflornithine), an irreversible inhibitor of ODC (5), exhibits notable efficacy against the central nervous system phase of African sleeping sickness caused by Trypanosoma brucei gambiense (3, 6). DFMO is also active against T. b. rhodesiense and T. congolense in murine models and has proven effective against other genera of protozoan parasites in vivo and in vitro, including Plasmodia (7), Giardia (8), and Leishmania (9). DFMO has been shown to induce a lethal polyamine depletion in both T. brucei (10) and L. donovani (9), the etiologic agent of visceral leishmaniasis, and toxicity to both species is ameliorated by polyamine addition (3, 9).The ability of trypanosomatids to undergo a very high frequency of homologous recombination allows the disruption of chromosomal loci with transfected drug resistance cassettes (11,12) and permits a direct test of gene function. This enables the creation of conditionally lethal parasite strains whose survival and ability to propagate are dependent upon the provision of compounds that can ameliorate the consequences of the genetic lesion. This genetic approach is predicated on the availability of c...

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Δarg), as well as wild-type and complemented Δarg controls (Δarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p &lt; 0.05); 2) poorer survival of Δarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Δarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Δarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Δarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Δarg parasites produced more IFN-γ and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.

Arginine is an essential amino acid for the human pathogen Leishmania but not to its host. Thus, the mechanism by which this protozoan parasite regulates cellular homeostasis of arginine is critical for its survival and virulence. In a previous study, we cloned and functionally characterized a high affinity arginine-specific transporter, LdAAP3, from Leishmania donovani. In this investigation, we have characterized the relationship between arginine transport via LdAAP3 and amino acid availability. Starving promastigotes for amino acids decreased the cellular level of most amino acids including arginine but also increased the abundance of both LdAAP3 mRNA and protein and up-regulated arginine transport activity. Genetic obliteration of the polyamine biosynthesis pathway for which arginine is the sole precursor caused a significant decrease in the rate of arginine transport. Cumulatively, we established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed. Our findings have led to the hypothesis that L. donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that regulates LdAAP3 expression and activity. Interestingly, this response of LdAAP3 to amino acid availability in L. donovani is identical to that of the mammalian cation amino acid transporter 1. Thus, we conjecture that Leishmania mimics the host response to amino acid availability to improve virulence.L-Arginine is a metabolically versatile cationic amino acid that provides a precursor function for the synthesis of a variety of bioactive molecules in all organisms. These molecules include nitric oxide, polyamines, phospho-and methylarginine, and proteins. Most of these molecules are essential for cell growth, and a few, like nitric oxide, are also used to protect hosts from pathogen invasion (1, 2). To enable simultaneous feeding of multiple and disparate biosynthetic pathways, cells must maintain ample cellular levels of arginine at all times. A variety of mechanisms for arginine homeostasis have been identified in mammals and fungi (3, 4). However, only very limited information on arginine homeostasis in protozoan parasites and its role in pathogenesis is available. Our laboratory aims to address these questions in the human pathogen Leishmania. Protozoan parasites of the genus Leishmania are the causative agents of a wide spectrum of human and veterinary diseases. Infections with Leishmania vary in their clinical manifestations depending upon the species ranging from self-healing skin lesions to visceral pathogenesis, which is invariably fatal if untreated (5). Leishmania species exhibit a digenetic life cycle that includes both extracellular promastigote and intracellular amastigote forms. The extracellular promastigotes develop in the alimentary tract of sand flies, whereas amastigotes reside within macrophage phagolysosomes (6, 7).Arginine is an essential amino acid for Leishmania (8), and therefore its metabolism and homeostasis dep...

Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal ⌬odc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the ⌬odc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent ⌬odc knockout lines were decreased ϳ80% compared to their wild-type counterpart. Furthermore, ␣-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both ⌬odc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the ⌬odc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania, has not been analyzed in L. donovani. To test ARG function in intact parasites, we generated Δarg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δarg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δarg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δarg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.