The Cloning and Characterization of Two ROP/RAC G-Proteins from Gossypium Hirsutum

Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation

The Cloning and Characterization of Two ROP/RAC G-Proteins from Gossypium Hirsutum

Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation

Engagement of Toll-like receptor-2 on cytotoxic T-lymphocytes occurs in vivo and augments antitumor activity

Toll-like receptors (TLRs) are among the fundamental molecules that alert the immune system to the presence of an infection by recognizing pathogen-associated molecules. Much of our understanding regarding TLR function stems from the study of innate immune cells. Recent studies by several groups, including ours, have shown that TLRs can function as costimulatory receptors for antigen-specific T cells, resulting in enhanced T-cell survival and increased expression of effector molecules. We report that the ligation of the TLR1/2 heterodimer on OT-1 cytotoxic T-lymphocytes (CTL) but not TLR2–/–OT-1 T cells increased cytolytic activity in vitro and in vivo. On the basis of these data, we tested the hypothesis that TLR1/2 stimulation on CTLs would enhance antitumor activity in a therapeutic model of B16-Ova melanoma. Adoptive OT-1 T-cell transfer into wild-type and MyD88–/– mice, followed by injection with TLR1/2 ligand, resulted in a synergistic antitumor effect, which correlated with the induction of CD8 T cells specific to various tumor antigens. In contrast, mice receiving TLR2–/–OT-1 T cells and TLR1/2 ligand showed minimal therapeutic efficacy. These findings emphasize the physiological significance of TLR2 engagement on CTLs and could make possible new approaches for the development of effective immunotherapies by manipulating TLR signaling within CTLs.—Asprodites, N., Zheng, L., Geng, D., Velasco-Gonzalez, C., Sanchez-Perez, L., Davila, E. Engagement of Toll-like receptor-2 on cytotoxic T-lymphocytes occurs in vivo and augments antitumor activity

When Toll-like receptor and T-cell receptor signals collide: a mechanism for enhanced CD8 T-cell effector function

Emerging reports reveal that activating Toll-like receptor-2 (TLR2)–MyD88 signals in CD8 T lymphocytes enhances cytokine production and cytotoxicity; however, the signaling pathway remains undefined. In the present study, we examined the physiologic significance and molecular mechanisms involved in this process. We found that TLR2 engagement on T-cell receptor transgenic CD8 OT-1 T cells increased T-bet transcription factor levels consequently, augmenting effector transcript and protein levels both in vivo and in vitro. In contrast, TLR2 agonist did not costimulate TLR2−/−OT-1 or MyD88−/−OT-1 T cells. Elevated T-bet levels in TLR2-MyD88–activated T cells was a consequence of increased biosynthesis resulting from the enhanced acti- vation of the mammalian target of the rapamycin (mTOR) pathway. Inhibiting mTOR, Akt, or protein kinase C in T cells abolished the costimulatory effects of the TLR2 agonist. In vivo, activating TLR2–MyD88 signals in T cells increased effector-molecule levels and enhanced the clearance of Listeria monocytogenes-Ova. These results help define a signaling pathway linking the TLR-MyD88 and mTOR pathway in an Akt- and protein kinase C–dependent manner. These results highlight a critical role for MyD88 signaling in T-cell activation and cytotoxicity. Furthermore, these findings offer the opportunity for improving the efficacy of vaccines and T cell–based immunotherapies by targeting TLR-MyD88 signaling within T cells