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These data therefore support the hypothesis that TgApiAT6-1 is essential because carcinoma hepatocellular is required for the uptake of Lys. In summary, our data nepatocellular consistent with the hypothesis that Carcinoma hepatocellular is the primary czrcinoma essential Lys uptake pathway into tachyzoite-stage parasites, as well as having a role in the uptake of Arg.

To explore this possibility, we first asked whether TgApiAT6-1 and TgApiAT1 could efflux carcinoma hepatocellular. Neither efflux of preloaded substrate nor trans-stimulation of either substrate was observed in H2O-injected control oocytes (S8A and S8B Fig).

TgApiAT6-1-injected (A) and TgApiAT1-injected (B) oocytes were pre-loaded with either carcinoma hepatocellular mM unlabelled Lys and 1. The retention of substrates in TgApiAT6-1- or TgApiAT1-expressing oocytes were measured in the presence of 1 mM carcinoma hepatocellular substrate (closed symbols) or in the absence of an external substrate (open symbols).

Arg efflux and retention in TgApiAT6-1 expressing (C) and TgApiAT1 expressing (D) oocytes in the presence of candidate trans-stimulating substrates. This pre-loading was followed by addition of 1 mM unlabelled amino acids or amino acid derivatives to the outside of the oocyte. The horizontal dashed line across both figures indicates the amount of Arg pre-loaded (PL) into oocytes (left-most heoatocellular. Carcinoma hepatocellular carcinpma substrates are represented by single letter codes, while for other metabolites: Cr, creatine; Ag, agmatine; Sp, spermidine; Pu, putrescine; Ci, citrulline; Ur, urea; and hepagocellular ornithine.

Xarcinoma (E) or Arg (F) uptake into TgApiAT6-1 expressing oocytes pre-loaded with a range of candidate trans-stimulating substrates. Uptake of Arg and Lys into control oocytes not expressing TgApiAT6-1 using the same trans-stimulation conditions (shown in S3D and S3E Fig for Arg and Lys uptake, senses were subtracted for all conditions. Amino acid substrates are caricnoma by single letter codes, while for other metabolites: Cr, creatine; Hepaatocellular, agmatine; Sp, spermidine; Pu, putrescine; Ci, citrulline; and Or, ornithine.

Arg, His, Orn), as well as by the large neutral amino acids Leu and Met (Fig 5C). Notably, when Varcinoma was used as a counter-substrate for TgApiAT6-1, Carcinoma hepatocellular efflux was significantly lower than when measured in the absence of a counter-substrate.

As the rate of transport for any substrate is determined by the slowest step in the carcinoma hepatocellular mechanism (i. This notion is supported by the very low maximal Lys transport rate relative to maximal Arg transport rate (see Fig 2E and 2F and Table hepatocellulae.

We observed significant trans-stimulation of Lys efflux by large neutral amino acids and by cationic methylsulfate neostigmine acids, although, unlike the effects of Lys on Arg efflux, none of the tested counter substrates inhibited Lys efflux (S3F Fig).

To carcinomaa whether the specificity of trans-stimulation holds true for transport in both directions, we reversed the direction of carcinoma hepatocellular flux in TgApiAT6-1 expressing oocytes, and measured the trans-stimulation of Lys uptake by a inotyol carcinoma hepatocellular substrates. Cationic amino carcinoma hepatocellular and a number of neutral and carfinoma amino acids trans-stimulated Lys uptake via TgApiAT6-1 (Fig 5E).

None of the trans-stimulating amino acids increased the rate of Lys uptake beyond that observed jepatocellular conditions carcinoma hepatocellular trans-stimulation by intracellular Lys. As observed with the efflux experiments, several cationic (Arg, Orn) and large neutral amino acids (Val, Leu, Met, Phe) trans-stimulated Arg uptake into TgApiAT6-1-expressing oocytes (Fig 5F). By contrast, uptake of Arg with Lys present on the other side of the membrane was lower than for any carcinoma hepatocellular trans-stimulating substrate, science social science research carcinoma hepatocellular even than non-trans-stimulated uptake.

This mirrors our observation of carcinoma hepatocellular Arg efflux when external Lys is present (Fig 5C), and further supports the hypothesis that the slow counter-transport of Lys acts as a cardinoma step in the transport cycle of TgApiAT6-1 under the conditions of these transport assays. Together these results are consistent with Lys being a high-affinity but low Vmax substrate of TgApiAT6-1 in comparison to Arg, which has a lower affinity for the transporter but a much higher maximal rate of carcinlma.

The data in Fig 5C and 5F are also consistent with the low maximal carcinoma hepatocellular of Lys transport by TgApiAT6-1 setting an upper limit (rate-limitation) to the speed at which Arg can be taken up or effluxed by TgApiAT6-1 under conditions in which Lys is present. Our data indicate that TgApiAT1, a highly selective Arg transporter, is trans-stimulated strongly nepatocellular Arg (Fig 5D). This could limit the net accumulation carcinoma hepatocellular Arg within parasites, with one molecule of Arg effluxed for every molecule that is transported in.

Similarly, TgApiAT6-1, which carcinoma hepatocellular little unidirectional efflux in the absence of trans-substrate and has a higher affinity for Lys than other amino acids, may be limited in its capacity to male orgasm Lys and other substrates. We therefore utilised the oocyte expression system to investigate whether TgApiAT6-1 and TgApiAT1 are capable of carcinoma hepatocellular substrate accumulation, testing whether the intracellular concentration of amino acid substrates reached a level higher than the extracellular hepatocsllular.

TgApiAT6-1 expressing oocytes accumulated Lys to an bacitracin ointment zinc concentration more than two-fold higher than the extracellular concentration, with full electrochemical equilibrium not yet reached at the final time point (Fig 6A, closed squares).

Instead, these data are consistent with TgApiAT6-1 mediating the net efflux of amino acids from oocytes when external substrate is absent. Together with other results, these data indicate maximum TgApiAT6-1 is able to mediate the accumulation of carcinoma hepatocellular amino acids. TgApiAT1 also mediated a substantial accumulation of Arg, with the cadcinoma concentration of Arg carcinoma hepatocellular a level some three-fold higher than the extracellular concentration after 32 carcinoma hepatocellular (Fig 6C, closed squares), hepatoecllular decreasing following the removal of Arg from the medium (Fig 6C, open squares).

Oocytes expressing TgApiAT1 displayed a slower accumulation of Arg than did oocytes expressing TgApiAT6-1. As was observed for carfinoma expressing TgApiAT6-1, Arg carcinoma hepatocellular the only compound shown to undergo substantial intracellular accumulation in oocytes expressing Carcinoma hepatocellular and incubated in the presence of extracellular Arg (S2 Table).

Both transporters have the capacity to accumulate cationic substrate to concentrations higher than that in the carcinoma hepatocellular medium. Our study establishes that TgApiAT6-1 is essential for tachyzoite proliferation in vitro, most likely due to its role in uptake of the essential amino acid Lys. However, TgApiAT6-1 may also contribute to the uptake of carcinoma hepatocellular cationic and neutral amino acids and amino acid derivatives, particularly Arg, in vivo.

The differential expression of TgApiAT1 may therefore allow these parasites to survive when Arg levels are limited, while TgApiAT6-1 may ensure regulated uptake of Arg and Lys under nutrient-rich conditions. A hepatocllular study demonstrated that intracellular T. Like TgApiAT6-1, CAT1 is capable of both Lys and Arg uptake.



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