Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

<div><p>RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoH^tg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoH^tg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4⁺CD8⁺ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2^-/-RhoH^tg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.</p></div

The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH^-/- mice.

<p>(A) Analysis of RhoH protein expression by western blot in RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes. (B, C) Analysis of RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4^-CD8^- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4⁺CD8⁺ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001</p

Analysis of T cell development in RhoH overexpressing mice.

<p>Analysis by flow cytometry of thymocytes and splenocytes from RhoH^+/+RhoH^Tg (red) and RhoH^+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4^-CD8^-CD44^-CD25⁺ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH^+/+RhoH^Tg and RhoH^+/+ mice in either MHC^+/+ (C, n = 6) or MHC^-/- (D, n = 5) background. Solid line and dashed line represent RhoH^+/+ and RhoH^+/+RhoH^Tg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4⁺ T (F) or CD8⁺ T (G) cells gated on TCRβ⁺ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.</p

Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.

<p>(A) FACS analysis of the <i>in vitro</i> differentiation of thymocytes to the DP stage. DN3 cells from Rag2^-/-, Rag2^-/-RhoH^tg, and Rag2^+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.</p

Bacilysocin, a Novel Phospholipid Antibiotic Produced by Bacillus subtilis 168

We have found a novel phospholipid antibiotic (named bacilysocin) which accumulates within (or associates with) the cells of Bacillus subtilis 168 and determined the structure by nuclear magnetic resonance and mass spectrometry analyses. The structure of bacilysocin elucidated was 1-(12-methyltetradecanoyl)-3-phosphoglyceroglycerol. Bacilysocin demonstrated antimicrobial activity, especially against certain fungi. Production of bacilysocin commenced immediately after growth ceased and before the formation of heat-resistant spores. The production of bacilysocin was completely blocked when the ytpA gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the ytpA gene did not significantly affect growth. Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected. Since the ytpA disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin