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Wang X.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Wu T.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Wu T.,Zhejiang University | Hu Y.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | And 6 more authors.
PLoS ONE | Year: 2012

Pno1 is a protein that plays a role in proteasome and ribosome neogenesis in yeast. So far, its functions in mammalian cells have not been investigated. To understand its function in mammals, we performed in situ hybridization analysis of Pno1 expression in different development stages and generated Pno1 gene knockout (KO) and transgenic (Tg) mice lineages. The results showed early lethality of homozygous Pno1 KO lineage caused, as demonstrated in parallel by ex vivo experiments, by arrest of embryo development before compaction stage. Though, heterozygous (HET) mice with 50% of normal Pno1 mRNA concentration were fertile and showed no obvious anomalies. The lymphoid organs of HET mice were normal in size, weight and cellularity, with normal T and B cell subpopulations. TCR-triggered activation and proliferation of HET T cells were normal. Proteasome activities in HET organs were uncompromised. Tg mice with actin promoter-driven Pno1 expression were also fertile, with no apparent anomalies, although they expressed 2-5-fold higher Pno1 mRNA levels. The lymphoid organs of Tg mice were of normal size, weight and cellularity with normal T and B cell sub-populations. TCR-triggered activation and proliferation of Tg T cells were normal. Tg organs and tissues presented normal proteasome activity as did their wild type counterparts. Tagged Pno1 over-expression in L cells and density gradient fractionation established that Pno1 existed in large complexes with sedimentation rates between 20S and 26S, bigger than mature 26S proteasomes. Pno1 in fractions did not coincide with 40S or 60S ribosome subunits. Our study indicates that Pno1 is essential for cellular functions, but only a small percentage of its normal level is sufficient, and excessive amounts are neither harmful nor useful. The nature of the large complexes it associates with remains to be identified, but it is certain that they are not mature proteasomes or ribosomes. © 2012 Wang et al. Source

Jin W.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Luo H.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame | Wu J.,Center Hospitalier Of Luniversite Of Montreal Crchum Hopital Notre Dame
Molecular Immunology | Year: 2014

The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function. © 2013 Elsevier Ltd. Source

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