Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 µM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-ß-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 µM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Assessment of total uncertainty in cocaine and benzoylecgonine wastewater load measurements.

To check the effectiveness of campaigns preventing drug abuse or indicating local effects of efforts against drug trafficking, it is beneficial to know consumed amounts of substances in a high spatial and temporal resolution. The analysis of drugs of abuse in wastewater (WW) has the potential to provide this information. In this study, the reliability of WW drug consumption estimates is assessed and a novel method presented to calculate the total uncertainty in observed WW cocaine (COC) and benzoylecgonine (BE) loads. Specifically, uncertainties resulting from discharge measurements, chemical analysis and the applied sampling scheme were addressed and three approaches presented. These consist of (i) a generic model-based procedure to investigate the influence of the sampling scheme on the uncertainty of observed or expected drug loads, (ii) a comparative analysis of two analytical methods (high performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry), including an extended cross-validation by influent profiling over several days, and (iii) monitoring COC and BE concentrations in WW of the largest Swiss sewage treatment plants. In addition, the COC and BE loads observed in the sewage treatment plant of the city of Berne were used to back-calculate the COC consumption. The estimated mean daily consumed amount was 107 ± 21 g of pure COC, corresponding to 321 g of street-grade COC.

Analysis of llicit and illicit drugs in waste, surface and lake water samples using large volume direct injection high performance liquid chromatography--electrospray tandem mass spectrometry (HPLC-MS/MS).

Llicit and illicit drugs represent a recent group of emerging contaminants and have been found in the aquatic environment. A HPLC-MS/MS method was developed using direct injection (DI) of larger volumes and a polar endcapped reversed-phase (RP) column to measure drug components in water samples belonging to the cocaine group, opiates, amphetamine-like stimulants and metabolites thereof. After validation, including sensitivity, linearity, recovery, precision and matrix effect studies, most drugs could be detected with limits of quantitation (LOQ) of 20 ng L(-1) in wastewater (WW) and 0.2 ng L(-1) in surface water. The major substances found in influents and effluents were cocaine (COC), benzoylecgonine (BE), morphine (MO), methadone (MD) and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) with concentrations up to 2 µg L(-1), followed by codeine (COD) and the amphetamines which ranged between 20 and 400 ng L(-1). Except for MO, COD and EDDP levels were generally lower in the effluents. River and lake water contained trace amounts of mainly BE, MD and EDDP from the high pg L(-1) to the low ng L(-1) level. Monitoring COC and BE levels over 11 consecutive days in influents and effluents suggests a consumption preference on week-end days. Finally, measuring an influent after a major music event revealed that sewage treatment plants (STPs) are exposed, for a limited period of time, to high concentration peaks of COC and BE as well as amphetamine-like stimulants such as ecstasy (MDMA).