Prevalence and Associated Risk Factors of Urinary Schistosomiasis among Primary School Pupils in the Jidawa and Zobiya Communities of Jigawa State, Nigeria.

Background: Urogenital schistosomiasis (UgS) is a parasitic disease caused by Schistosoma haematobium and can lead to chronic ill-health. Nigeria is endemic for schistosomiasis, but epidemiology of UgS has not been studied in most states. This study was conceived with the aim to contribute towards an accurate national picture of UgS in Nigeria. The prevalence of UgS and the associated risk factors were for the first time investigated among primary school pupils in Jidawa and Zobiya communities of the Dutse Local Government Area (LGAs) of Jigawa State, Nigeria. Method: Focus group discussions with teachers and parents were conducted. After obtaining written consent from parents, questionnaires were administered to pupils to obtain socio-demographic data and information on water contact activities. Urine samples (279) were collected and processed by the urine filtration technique to evaluate haematuria and the presence of S. haematobium eggs. Results: Prevalences of 65.7% (90/137) and 69.0% (98/142) were recorded in the Jidawa and Zobiya communities, respectively. In both communities, there was a significant association between gender and UgS: 63.3% of the infected pupils were males as compared to 36.7% females (χ2 = 5.42, p = 0.020). Grade 5 students had a significantly higher prevalence (χ2 = 17.919, p = 0.001) (80.0%) compared to those in grades 2, 3, 4, and 6 (63.8%, 66.7%, 61.5%, and 64.6%, respectively). Water contact activities showed that pupils involved in fishing, irrigation, and swimming were at greater risk of becoming infected in Jidawa and Zobiya, with odds ratios (risk factors) of 5.4 (0.994-28.862) and 4.1 (1.709-9.862), respectively (p = 0.05). Conclusion: Both the Jidawa and Zobiya communities of the Dutse LGAs of Jigawa State are hyperendemic for UgS. In collaboration with the State Ministry of Health, mass administration of praziquantel was carried out in the Jidawa and Zobiya communities after this study.

Effects of some natural leads on Trypanosoma and Leishmania strains.

Well-known medical herbal compounds including apigenin, daidzein, phyllanthin and tyramine were assessed against Trypanosoma and Leishmania protozoans. Two strains of the bloodstream forms of Trypanosoma brucei: s427-WT and TbAT1-B48, and Leishmania major and Leishmania mexicana promastigotes were utilised. Among selected natural compounds, apigenin and daidzein displayed moderate activity against African trypanosomes with EC50 16 µM for wild-type sensitive control strain. Tyramine was not found to be very active for trypanosomes strains while all compounds were found to have trivial activity for the inhibition of Leishmania mexicana strains.

Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters.

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

Chemotherapeutic strategies against Trypanosoma brucei: drug targets vs. drug targeting.

Trypanosoma brucei rhodesiense and T. b. gambiense are the causative agents of sleeping sickness, a fatal disease that affects 36 countries in sub-Saharan Africa. Nevertheless, only a handful of clinically useful drugs are available. These drugs suffer from severe side-effects. The situation is further aggravated by the alarming incidence of treatment failures in several sleeping sickness foci, apparently indicating the occurrence of drug-resistant trypanosomes. Because of these reasons, and since vaccination does not appear to be feasible due to the trypanosomes' ever changing coat of variable surface glycoproteins (VSGs), new drugs are needed urgently. The entry of Trypanosoma brucei into the post-genomic age raises hopes for the identification of novel kinds of drug targets and in turn new treatments for sleeping sickness. The pragmatic definition of a drug target is, a protein that is essential for the parasite and does not have homologues in the host. Such proteins are identified by comparing the predicted proteomes of T. brucei and Homo sapiens, then validated by large-scale gene disruption or gene silencing experiments in trypanosomes. Once all proteins that are essential and unique to the parasite are identified, inhibitors may be found by high-throughput screening. However powerful, this functional genomics approach is going to miss a number of attractive targets. Several current, successful parasiticides attack proteins that have close homologues in the human proteome. Drugs like DFMO or pyrimethamine inhibit parasite and host enzymes alike--a therapeutic window is opened only by subtle differences in the regulation of the targets, which cannot be recognized in silico. Working against the post-genomic approach is also the fact that essential proteins tend to be more highly conserved between species than non-essential ones. Here we advocate drug targeting, i.e. uptake or activation of a drug via parasite-specific pathways, as a chemotherapeutic strategy to selectively inhibit enzymes that have equally sensitive counterparts in the host. The T. brucei purine salvage machinery offers opportunities for both metabolic and transport-based targeting: unusual nucleoside and nucleobase permeases may be exploited for selective import, salvage enzymes for selective activation of purine antimetabolites.

A diminazene-resistant strain of Trypanosoma brucei brucei isolated from a dog is cross-resistant to pentamidine in experimentally infected albino rats.

Trypanosomosis is a major cause of mortality for dogs in Nigeria and treatment with diminazene aceturate has steadily become less effective, either as a result of low quality of the locally available diminazene preparations or of drug resistance. To investigate these alternatives, samples of locally obtained drugs were analysed for diminazene aceturate content and a strain of Trypanosoma brucei brucei was isolated from a diminazene-refractory dog in Nsukka, south-eastern Nigeria, and used to infect albino rats. The quality of diminazene aceturate-based preparations was variable, with two preparations containing less than 95% of the stated active compound. Rats infected with T. brucei isolated from the dog were treated 7 and 10 days after infection either with 7 mg/kg diminazene aceturate (intraperitoneally, once) or with 4 mg/kg pentamidine isethionate (intramuscularly, 7 consecutive days). Relapse rates were 100% for both trypanocides in the groups of rat treated 10 days post-infection, and 83% and 50% of rats treated 7 days after infection relapsed to diminazene aceturate and pentamidine isethionate, respectively. Careful consideration of physiological parameters showed that pentamidine was only marginally superior to diminazene aceturate as applied in this study. It was concluded that dogs in Nigeria are infected with genuinely diminazene aceturate-resistant trypanosomes that appear to be cross-resistant to pentamidine isethionate.

Identification of the first pyrimidine nucleobase transporter in Leishmania: similarities with the Trypanosoma brucei U1 transporter and antileishmanial activity of uracil analogues.

While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0.32+/-0.07 microM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.

Selective transport of a new class of purine antimetabolites by the protozoan parasite Trypanosoma brucei.

Purine antimetabolites have been very successful therapeutic agents against a host of infectious diseases and malignancies. Success of the treatment relies as much on the efficient accumulation by the target cell or organism as it does on selective action on a vital biochemical pathway of the target cell. Here we compare the ability of a new class of tricyclic purine antimetabolites to interact with transporters from human erythrocytes or Trypanosoma brucei. We show that these compounds display a remarkable selectivity for the parasite's transporters. The adenine analogue showed greater trypanocidal activity than the hypoxanthine or guanine analogues in vitro.

Uptake of pentamidine in Trypanosoma brucei brucei is mediated by the P2 adenosine transporter and at least one novel, unrelated transporter.

Diamidine drugs such as pentamidine and berenil (diminazene aceturate) are vital drugs for the treatment of early stage human African trypanosomiasis and the corresponding veterinary condition, respectively. The action of diamidines on trypanosomes is critically dependent on their efficient uptake by the parasite. We have therefore investigated the mode of uptake of pentamidine by Trypanosoma brucei brucei, using [(125)I]iodopentamidine as a permeant. [(125)I]Iodopentamidine uptake was linear for up to 15 min and inhibited by adenosine with a K(i) value of 0.64+/-0.03 microM to a maximum of 50-70%. The adenosine-sensitive flux was also inhibited by adenine with a K(i) value of 0.44+/-0.04 microM. Iodopentamidine uptake was saturable, with the adenosine-insensitive flux displaying a K(m) of 22+/-2 microM and a V(max) of 2.2+/-0.9 pmol(10(7) cells)(-1)s(-1), whereas the adenosine-sensitive flux was inhibited by much lower iodopentamidine concentrations. These results clearly demonstrate that iodopentamidine is taken up by at least two different T. b. brucei transporters, an adenosine-sensitive pentamidine transporter (ASPT1) and a low-affinity pentamidine transporter (LAPT1). The identity of these transporters was investigated, and their significance for drug uptake and resistance in African trypanosomes is discussed.

Transporters in African trypanosomes: role in drug action and resistance.

Sleeping sickness is an increasing problem in many parts of sub-Saharan Africa. The problems are compounded by the lack of new medication, and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. Over the last few years, much progress has been made in understanding how drug action, and the development of resistance, is related to the mechanisms by which the parasite ingests the drugs. In some cases novel transporters have been identified. In other cases, transporters do not appear to be involved in drug uptake, and selectivity must lie with other parasite features, such as a specific target or activation of the drug. Lessons learned from studying the uptake of drugs currently in use may assist the design of a much needed new generation of trypanocides.

Uptake of pentamidine in Trypanosoma brucei brucei is mediated by three distinct transporters: implications for cross-resistance with arsenicals.

The trypanocidal action of pentamidine is dependent on the rapid, selective accumulation of this drug by the parasite. We have investigated pentamidine transport by the bloodstream and procyclic life cycle stages of Trypanosoma brucei brucei. In bloodstream forms, 50 to 70% of [(3)H]pentamidine was transported by an adenosine-sensitive pentamidine transporter (ASPT1) that displayed a K(m) value of 0.26 +/- 0.03 microM and K(i) values of 0.45 +/- 0.04 and 2.5 +/- 0.8 microM for adenine and berenil, respectively. These values are very similar to those for inhibition of [(3)H]adenosine uptake by the P2 adenosine/adenine transporter, suggesting that ASPT1 and P2 may be identical. The remaining 30 to 50% of [(3)H]pentamidine transport was mediated by a low-capacity high-affinity pentamidine transporter (HAPT1) and a high-capacity low-affinity pentamidine transporter (LAPT1), with K(m) values of 36 +/- 6 nM and 56 +/- 8 microM, respectively. HAPT1 was inhibited by propamidine but displayed only low affinity to berenil and stilbamidine, whereas LAPT1 was not inhibited by any of these diamidines. Neither transporter was inhibited by melarsen oxide. In procyclics, an HAPT1-analog (procyclic pentamidine transporter; PPT1) was characterized, but no adenosine-sensitive pentamidine transport could be detected. Treatment with ionophores revealed that PPT1 may be a proton/pentamidine cotransporter.

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei.

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.

Adenosine transporters in bloodstream forms of Trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs.

Adenosine influx by Trypanosoma brucei brucei P1 and P2 transporters was kinetically characterized. The P1 transporter displayed a higher affinity and capacity for adenosine (K(m) = 0.38 +/- 0.10 microM, V(max) = 2.8 +/- 0.4 pmol x 10(7) cells(-1) x s(-1)) than the P2 transporter (K(m) = 0.92 +/- 0.06 microM, V(max) = 1.12 +/- 0.08 4 pmol x 10(7) cells(-1) x s(-1)). To formulate a structure-activity relationship for the interaction of adenosine with the transporters, a series of analogs were evaluated as potential inhibitors of adenosine transport, and the K(i) values were converted to binding energy. The P1 transporter was found to be selective inhibited by purine nucleosides (K(i) approximately 1 microM for inosine and guanosine), but nucleobases and pyrimidines had little effect on P1-mediated transport. The P1 transporter appears to form hydrogen bonds with N3 and N7 of the purine ring as well as with the 3' and 5' hydroxyl groups of the ribose moiety, with apparent bond energies of 12.8 to 15.8 kJ/mol. The P2 transporter, in contrast, had high-affinity (K(i) = 0.2-4 microM) for 6-aminopurines, including adenine, 2'-deoxyadenosine, and tubercidin, but not for any oxopurines. The main interaction of adenosine with the P2 transporter is suggested to be via hydrogen bonds to N1 and the 6-amino group. Additional pi-pi interactions of the purine ring and electrostatic interactions with N9 may also be important. The predicted substrate recognition motif of P2, but not of P1, corresponds to parts of the melaminophenylarsenical and diamidine molecules, confirming the potent inhibition observed with these trypanocides for P2-mediated adenosine transport (K(i) = 0.4-2.4 microM).

Characterization of a nucleoside/proton symporter in procyclic Trypanosoma brucei brucei.

Adenosine transport at 22 degrees C in procyclic forms of Trypanosoma brucei brucei was investigated using an oil-inhibitor stop procedure for determining initial rates of adenosine uptake in suspended cells. Adenosine influx was mediated by a single high affinity transporter (Km 0.26 +/- 0.02 microM, Vmax 0.63 +/- 0.18 pmol/10(7) cells s-1). Purine nucleosides, with the exception of tubercidin (7-deazaadenosine), and dipyridamole inhibited adenosine influx (Ki 0.18-5.2 microM). Purine nucleobases and pyrimidine nucleosides and nucleobases had no effect on adenosine transport. This specificity of the transporter appears to be similar to the previously described P1 adenosine transporter in bloodstream forms of trypanosomes. Uptake of adenosine was Na+-independent, but ionophores reducing the membrane potential and/or the transmembrane proton gradient (monitored with the fluorescent probes bis-(1,3-diethylthiobarbituric acid)-trimethine oxonol and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, respectively) inhibited adenosine transport. Similarly, an increase in extracellular pH from 7.3 to 8.0 reduced adenosine influx by 30%. A linear correlation was demonstrated between the rate of adenosine transport and the protonmotive force. Adenosine uptake was accompanied by a proton influx in base-loaded cells and was also shown to be electrogenic. These combined results indicate that transport of adenosine in T. brucei brucei procyclics is protonmotive force-driven and strongly suggest that the adenosine transporter functions as an H+ symporter.

A highly selective, high-affinity transporter for uracil in Trypanosoma brucei brucei: evidence for proton-dependent transport.

The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier.

Purine nucleobase transport in bloodstream forms of Trypanosoma brucei is mediated by two novel transporters.

The mechanism and inhibitor sensitivity of hypoxanthine transport by bloodstream forms of Trypanosoma brucei brucei was investigated. The dose response curve for the inhibition of hypoxanthine transport (1 microM) by guanosine was biphasic; approximately 90% of transport activity was inhibited with a Ki value of 10.8 +/- 1.8 microM, but 10% of the activity remained insensitive to concentrations as high as 2 mM. These two components of hypoxanthine transport are defined as guanosine-sensitive (H2) and guanosine-insensitive (H3). Hypoxanthine influx by both components was saturable, but there was a marked difference in their Km values (123 +/- 15 nM and 4.7 +/- 0.9 microM for H2 and H3, respectively) although the Vmax values (1.1 +/- 0.2 and 1.1 +/- 0.1 pmol (10[7] cells)[-1] s[-1], n = 3) were similar. Hypoxanthine uptake via the H2 carrier was inhibited by purine bases and analogues as well as by some pyrimidine bases and one nucleoside (guanosine), whereas the H3 transporter was sensitive only to inhibition by purine nucleobases. H2-mediated hypoxanthine uptake was inhibited by ionophores, ion exchangers and the potential H+-ATPase inhibitors, N,N'-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM). Measurements of the intracellular pH and membrane potential of bloodstream trypanosomes in the presence and absence of these agents established a linear correlation between protonmotive force and rate of [3H]hypoxanthine (30 nM) uptake. We conclude that hypoxanthine transport in bloodstream forms of T. b. brucei occurs by two transport systems with different affinities and substrate specificities, one of which, H2, appears to function as a H+-/hypoxanthine symporter.

Hypoxanthine uptake through a purine-selective nucleobase transporter in Trypanosoma brucei brucei procyclic cells is driven by protonmotive force.

The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22 degrees C was rapid and saturable, exhibiting an apparent Km of 9.3 +/- 2.0 microM and a Vmax of 4.5 +/- 0.8 pmol x (10(7) cells)(-1) x s(-1). All the natural purine nucleobases tested (Ki 1.8-7.2 microM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi), the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH 7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electrogenic. These data show that hypoxanthine uptake in T. b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.

The secretion of alpha-MSH from xenopus melanotropes involves calcium influx through omega-conotoxin-sensitive voltage-operated calcium channels.

The secretory activity of endocrine cells largely depends on the concentration of free cytosolic calcium. We have studied the mechanisms that are involved in supplying the calcium necessary for the secretion of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells in the pituitary intermediate lobe of the amphibian Xenopus laevis. Using whole-cell voltage clamp, high-voltage activated calcium currents were observed, with a peak current between 0 and +20 mV. Two types of Ca(2+)-currents appeared, depending on the experimental setup. An inactivating current, which was observed after a 10 msec depolarizing prepulse, resembled currents through N-type channels as it was clearly inhibited by 1 microM omega-conotoxin. The second type was a non-inactivating current, which was blocked up to 50% by 1 microM nifedipine, indicating its L-type nature. Only a small component of this inactivating current could be blocked by omega-conotoxin. No evidence was found for the presence of transient, low-voltage activated currents. The spontaneous secretion of alpha-MSH from superfused neurointermediate lobes was dependent on extracellular calcium, as low calcium conditions (10(-4)-10(-8) M) rapidly inhibited this process. Under these conditions, secretion was not affected by depolarizing concentrations of potassium chloride. The calcium ionophore A23187 increased secretion under low calcium conditions, but had no effect on spontaneous alpha-MSH release. These results suggest that spontaneous alpha-MSH release depends on influx of calcium through voltage-operated calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)