The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for hostparasite signaling.

Women develop certain autoimmune diseases more often than men. It has been hypothesized that this may relate to the development of more robust T-helper (Th)1 responses in women. To test whether women exhibit a Th1 bias, we isolated naïve cluster of differentiation (CD)4 + T cells from peripheral blood of healthy women and men and measured the proliferation and cytokine production by these cells in response to submaximal amounts of anti-CD3 and anti-CD28. We observed that CD4 + T cells from women produced higher levels of IFNγ as well as tended to proliferate more than male CD4 + T cells. Intriguingly, male CD4 + T cells instead had a predilection toward IL-17A production. This sex dichotomy in Th cytokine production was found to be even more striking in the Swiss/Jackson Laboratory (SJL) mouse. Studies in mice and humans indicated that the sexual dimorphism in Th1 and Th17 cytokine production was dependent on the androgen status and the T-cell expression of peroxisome proliferator activated receptor (PPAR)α and PPARγ. Androgens increased PPARα and decreased PPARγ expression by human CD4 + T cells. PPARα siRNA-mediated knockdown had the effect of increasing IFNγ by male CD4 + T cells, while transfection of CD4 + T cells with PPARγ siRNAs increased IL-17A production uniquely by female T cells. Together, our observations indicate that human T cells exhibit a sex difference in the production of IFNγ and IL-17A that may be driven by expressions of PPARα and PPARγ. autoimmunity | cytokines | experimental autoimmune encephalomyelitis | gender | nuclear receptor

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The 3-unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide undergoes a unique N-O bond cleavage to the active ␣-cyanoenol metabolite A771726, which resides in the same oxidation state as the parent. In vitro studies were conducted to characterize drug-metabolizing enzyme(s) responsible for ring opening and to gain insight into the mechanism of ring opening. Under physiological conditions, leflunomide was converted to A771726 in rat and human plasma (rat plasma, t 1/2 ‫؍‬ 36 min; human plasma, t 1/2 ‫؍‬ 12 min) and whole blood (rat blood, t 1/2 ‫؍‬ 59 min; human blood, t 1/2 ‫؍‬ 43 min). Human serum albumin also catalyzed A771726 formation, albeit at a much slower rate (t 1/2 ‫؍‬ 110 min). Rat and human liver microsomes also demonstrated NADPH-dependent A771726 formation (human liver microsomes, V max ‫؍‬ 1,797 pmol/min/mg and K m ‫؍‬ 274 M). Leflunomide metabolism in microsomes was sensitive to furafylline treatment, suggesting P4501A2 involvement. 3-Methylleflunomide, which contained a 3-methyl substituent on the isoxazole ring, was resistant to ring opening in base, plasma, blood, and liver microsomes. In microsomes, two monohydroxylated metabolites were formed, and metabolite identification studies established the 3-and the 5-methyl groups on the isoxazole ring as sites of hydroxylation. These results indicate that the C3-H in leflunomide is essential for ring opening. Although A771726 formation in human liver microsomes or recombinant P4501A2 required NADPH, its formation was greatly reduced by oxygen or carbon monoxide, suggesting that the isoxazole ring opening was catalyzed by the P450Fe(II) form of the enzyme. A mechanism for the P450-mediated ring scission is proposed in which the isoxazole ring nitrogen or oxygen coordinates to the reduced form of the heme followed by charge transfer from P450Fe(II) to the C‫؍‬N bond or deprotonation of the C3-H, which results in a cleavage of the N-O bond.

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structures of the 96-nm repeat from Trypanosoma brucei, a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in T. brucei reflects adaptations to support unique motility needs in diverse host environments.

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