rather than the primary duct span utilizing the length way of measuring feature by the complete mammary gland

Taken together, these data implicate Albumax II as the labile factor in fresh Albumax-complete RPMI that influences the potency of pantothenamides. The activity of pantetheinases, enzymes that catalyze the hydrolysis of pantetheine to pantothenate and cysteamine, has previously been detected in human serum. Since pantetheine is a secondary amide of pantothenate it more closely resembles the pantothenamides than pantothenol and other previously reported antiplasmodial pantothenate analogues do. In light of this, we considered the possibility that pantothenamides in this series are pantetheinase substrates and that Albumax II may be a source of pantetheinase activity. To explore this hypothesis we adapted a fluorescence-based assay used previously to measure Nacetyl- 1-D-myo-inosityl-2-amino-2-deoxy-a-D-glucopyranoside deacetylase activity to measure any breakdown of compound 12 to pantothenate and isobutylamine. This assay utilizes fluorescamine, a molecule which is itself non-fluorescent, but generates a fluorescent product upon reaction with primary amines. Using an isobutylamine standard curve, fluorescence measurements were converted to isobutylamine concentrations. As shown in Figure 7, when compound 12 was incubated with recombinant human pantetheinase, primary amine was generated, and the concentration increased approximately linearly with time before reaching a maximum after,4 h. The maximum reached corresponded to an isobutylamine concentration of,200 mM, consistent with all of the Tissues would therefore be a really useful advancement for the treatment method of EDD pantothenamide present having been hydrolyzed. By contrast, pantothenamide hydrolysis was not observed when compound 12 was incubated under the same conditions in the absence of pantetheinase. Hence, these data are consistent with pantetheinase mediating the hydrolysis of pantothenamides in addition to pantetheine. Primary amine was also generated during incubation of compound 12 with Albumax II, consistent with Albumax II mediating hydrolysis of the pantothenamide. Furthermore, incubation of Albumax II in the absence of pantothenamide did not result in primary amine generation, which eliminated the possibility that the amine generated resulted from degradation of the Albumax II and not the pantothenamide. In the presence of Albumax II, over one third of the pantothenamide present was hydrolyzed during a 6 h incubation, and pantothenamide hydrolysis appeared to reach completion within 24 h. To establish whether Albumax II-mediated pantothenamide hydrolysis could explain the reduced potency of pantothenamides in fresh Albumax-complete RPMI, we compared the activity of compound 12 in aged Albumax-complete RPMI with and without recombinant human pantetheinase. As shown in Figure 8, 100 ng/mL recombinant human pantetheinase alleviated the antiplasmodial effect of compound 12 in aged Albumax-complete RPMI. This result is consistent with pantetheinase-mediated pantothenamide degradation occurring under in vitro culture conditions and, in turn, attenuating the antiplasmodial effect of pantothenamides. Importantly, the activity of compound 12 in aged Albumax-complete RPMI supplemented with recombinant human pantetheinase increased after the medium was incubated at 37uC for 40 h. Moreover, the pantothenamide was more potent in this medium than in aged Albumax-complete medium incubated in parallel but to which the recombinant human pantetheinase was added only after the second incubation at 37uC. The latter provides evidence that the increase in pantothenamide activity is largely due to inactivation of pantetheinase and not a result of further inactivation of an independent component of the medium. Taken together these data demonstrate that the antagonizing effect of pantetheinase in parasite culture medium can be alleviated by incubating the medium at 37uC, and are consistent with inactivation of pantetheinase occurring during the incubation. That pantothenamides possess antibacterial activity has been known for some time. In this study we show for the first time that pantothenamides also possess antiplasmodial activity. Additionally, we show there to be a labile serum-derived factor common to Albumax II and human serum that specifically antagonizes the antiplasmodial activity of pantothenamides and thereby masks their potency.

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