All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine͞pyrimi-dine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Parasitic protozoa of the genus Leishmania are the etiological agents of leishmaniasis, a disease that affects an estimated 12 million people worldwide (1) and ranges from the disfiguring cutaneous form to fatal visceral leishmaniasis (2). Because current empirically identified drugs suffer from many deficiencies, including toxicity and resistance, it is important to identify unique biochemical targets that could be exploited for rational development of improved therapies. Perhaps the most striking metabolic discrepancy between parasites and their hosts is the purine pathway. Whereas most mammalian cells synthesize purines de novo, all parasitic protozoa studied to date are unable to synthesize purines (3) and consequently must rely on purine acquisition from their hosts for survival and growth. The first step in this salvage pathway involves the transport of these substrates across the parasite plasma membrane. Moreover, these purine transporters initiate the uptake of certain pyrazolopyrimidine analogs of hypoxanthine and inosine that are toxic to both Leishmania and Trypanosoma (4). These pyrazolopyrimidines, such as allopurinol, allopurinol riboside, and formycin B, are subsequently metabolized to the nucleotide level by the parasite metabolic machinery and incorporated into RNA, metabolic transformations that do not occur in mammalian cells (4). Both the essential nutritional function of these transporters and their roles in mediating the toxicities of well-characterized antiparasitic agents provide compelling rationale to study these membrane permeases at the molecular level.Biochemical and genetic studies have established that Leishmania donovani parasites express two distinct nucleoside transporters with nonoverlapping substrate specificities (5). One transporter mediates the uptake of adenosine and pyrimidine nucleosides and also transports tubercidin, a cytotoxic analog of adenosine, whereas the other transporter allows membrane permeation of guanosine, inosine, and formycin B (5). Parasites deficient in either or both transport activities have been isolated by mutagenesis with N-methyl-NЈ-nitro-N-nitrosoguanidine followed by selection in tubercidin or formycin B (6). The availability of these null mutants provided a functional strategy for cloning genes encoding each of these nucleoside permeases.In the present study, we have transfected the adenosine͞pyrimidine nucleoside transport-deficient TUBA5 cell line with a cosmid library containing inserts of L. donovani genomic DNA (7) and screened individual transfectants for restoration of tubercidin...

PURINE TRANSPORT AND SALVAGE IN PARASITIC PROTOZOAOne distinctive feature of the biochemistry of parasitic protozoa is their absolute reliance upon the salvage of preformed purines from their vertebrate and invertebrate hosts. While many mammalian cells possess the innate ability to synthesize purines de novo, all protozoa so far examined that exhibit a parasitic life style lack these biosynthetic pathways and have therefore elaborated a variety of salvage pathways ( Fig. 1) that enable them to acquire preformed purines from their hosts (14). These nutrients are imported from the host as either nucleosides or nucleobases, either of which can serve as a purine source for the parasite. The first step in these salvage pathways entails the uptake of the purine nucleosides or nucleobases from the host milieu and is mediated by various nucleoside or nucleobase transporters, located in the plasma membrane of the parasite, that provide substrate-specific permeation routes. While the enzymes of purine salvage have been studied in considerable molecular detail (7), the identification of individual purine transporters at the molecular level is a more recent development. However, over the past several years a plethora of genes encoding nucleoside or nucleobase permeases from parasitic protozoa have been identified and functionally expressed. The increasing understanding of these carriers at the molecular level provides an appropriate setting for a timely review of these important transporters and a comparison of their properties to related and distinct purine permeases of other organisms. This article will concentrate primarily on purine transporters from the Kinetoplastid parasites Leishmania and Trypanosoma brucei, since these are the protozoa with which the majority of the studies have been accomplished. However, we will also briefly discuss related work on transporters from the Apicomplexa Plasmodium falciparum and Toxoplasma gondii. This review does not attempt to be exhaustive but focuses rather upon recent results utilizing molecular genetic approaches.A principal reason for the interest in purine transport in these protozoa is the essential nature of purine salvage for this large family of parasites. Thus, while some permeases may provide nutrients that are nonessential, albeit advantageous, to the fitness of the organism in its natural environment, the purine transporters as a group are likely to be required for parasite viability in all life cycle stages. A second compelling reason to study these transporters is that they also mediate the uptake of a variety of cytotoxic drugs, many, but not all, of which are purine homologs (11,13,49). Consequently, the import of subversive substrates that are metabolized, often uniquely, by parasite purine salvage enzymes is absolutely dependent upon the purine permeases. Thus, this family of permeases plays an important role in the delivery of drugs or experimental therapeutic compounds, and mutations in these carriers can cause drug resistance (5, 13, 36). STUDIES OF PURINE TR...

Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 reestablished their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K m ‫؍‬ 0.3 M) and guanosine (K m ‫؍‬ 1.7 M) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.Leishmania donovani is a protozoan parasite and the etiologic agent of visceral leishmaniasis, a devastating and invariably fatal disease if untreated. The parasite exhibits an intricate life cycle in which the extracellular, flagellated promastigote exists in the phlebotomine sandfly vector, and the intracellular amastigote resides in the phagolysosome of macrophages and other reticuloendothelial cells of the mammalian host. Drugs are the only defense against visceral leishmaniasis, but the efficacy of these empirically derived agents is compromised both by drug toxicity and resistance (1). Thus, it is increasingly imperative to identify new and unique biochemical targets in the parasite for potential therapeutic exploitation.Among the most conspicuous metabolic differences between parasites and their mammalian hosts is the purine pathway. Whereas animal cells synthesize purine nucleotides de novo, all protozoan parasites are incapable of synthesizing purines and depend upon purine acquisition from their hosts to survive and proliferate (2). Hence, each genus of parasite has evolved a unique complement of purine salvage enzymes in order to scavenge purines from the host milieu (2). The first step in this salvage process involves the translocation of purines across the parasite plasma membrane, a process mediated by membrane permeases. These permeases also initiate the uptake of pyrazolopyrimidine nucleobase and nucleoside analogs of hypoxanthine and inosine that are selectively toxic to Leishmania spp. (3, 4). Thus, purine transporters play vital roles in both purine nutrition and antiparasitic drug targeting i...

Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH) 3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH) 3 and CH 3 As(OH) 2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH) 3 and CH 3 As (OH) 2 . The K m of GLUT1 is to 1.2 mM for CH 3 As(OH) 2 , compared to a K m of 3 mM for glucose. Inhibition between glucose and CH 3 As(OH) 2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH) 3 and CH 3 As(OH) 2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.Arsenic ranks first on the United States Government's Comprehensive Environmental Response, Compensation, and Liability (Superfund) Act Priority List of Hazardous Substances . In response to the Safe Water Drinking Act, the United States Environmental Protection Agency has set the allowable level of arsenic in drinking water at 10 ppb , which is less than the level in the water supplies of many U.S. municipalities . In addition, the Environmental Protection Agency's Office of Pesticide Programs is concerned with exposure to the organic arsenicals methylarsenic acid and dimethylarsenic acid, pentavalent arsenicals used as pesticides and herbicides .Health effects associated with arsenic exposure include cardiovascular and peripheral vascular disease, neurological disorders, diabetes mellitus and various cancers, including liver, bladder, kidney and skin [1][2][3]. Arsenic trioxide, which is As 2 O 3 in the solid, anhydrous form and As (OH) 3 in solution at physiological pH [4], is used clinically as a chemotherapeutic drug for treatment of acute promyelocytic leukemia [5]. Although epidemiological studies demonstrated that arsenic exposure is associated with a high frequency of circulatory and neurological problems [2,6,7], how arsenic causes non-cancer-related diseases is far from clear. Here we report that GLUT1 facilitates uptake of As(OH) 3 and CH 3 As(OH) 2 using heterologous expression in S. cerevisiae and X. laevis öocytes. S. cerevisiae is a valuable model system for structure-function studies of mammalian membrane proteins such as GLUT1 [22,23]. A yeast strain with low uptake of As(OH) 3 and CH 3 As(OH) 2 was constructed by deletion of all 18 hexose transporters, FPS1, which encodes an aquaglyceroporin that facilitates As(OH) 3 influx, and ACR3, the gene for an...

Growth hormone (GH) insensitivity syndrome (GHIS) is a rare clinical condition in which production of insulin-like growth factor 1 is blunted and, consequently, postnatal growth impaired. Autosomal-recessive mutations in signal transducer and activator of transcription (STAT5B), the key signal transducer for GH, cause severe GHIS with additional characteristics of immune and, often fatal, pulmonary complications. Here we report dominant-negative, inactivating STAT5B germline mutations in patients with growth failure, eczema, and elevated IgE but without severe immune and pulmonary problems. These STAT5B missense mutants are robustly tyrosine phosphorylated upon stimulation, but are unable to nuclear localize, or fail to bind canonical STAT5B DNA response elements. Importantly, each variant retains the ability to dimerize with wild-type STAT5B, disrupting the normal transcriptional functions of wild-type STAT5B. We conclude that these STAT5B variants exert dominant-negative effects through distinct pathomechanisms, manifesting in milder clinical GHIS with general sparing of the immune system.