The liverwort Marchantia polymorpha L. is an important model species for investigating land plant evolution. Effective genetic transformation techniques are crucial for plant molecular biology and simplified or improved techniques for specific cultivars or strains can accelerate research. Over the past several years, we developed a simple Agrobacterium-mediated transformation technique for M. polymorpha named AgarTrap (Agar-utilized transformation with pouring solutions). AgarTrap is an easy technique that involves pouring the appropriate solutions onto plant materials on a single solid plate of medium. We recently improved AgarTrap using gemmalings (G-AgarTrap) of the M. polymorpha female model strain BC3-38 and achieved a transformation efficiency of nearly 100%. Based on this improved technique, in the current study, we adopted two factors (sealing the Petri dish with Parafilm and dark treatment during co-cultivation) and optimized two factors (Agrobacterium strain and pre-culture period) of the improved G-AgarTrap for other model strains of M. polymorpha, the male strain Takaragaike-1 (Tak-1) and the female strain Takaragaike-2 (Tak-2). After optimization, the transformation efficiency of Tak-1 using G-AgarTrap was as high as 55% compared to approximately 30% using the previous protocol. Furthermore, using Tak-2, we achieved a transformation efficiency of nearly 100%. Our improved G-AgarTrap technique for Tak-1 and Tak-2 represents a promising tool for promoting the study of Marchantia.

2013-02-01·Current medicinal chemistry2区 · 医学

4-Mer Hyaluronan Oligosaccharides Stimulate Inflammation Response in Synovial Fibroblasts in Part via TAK-1 and in Part via p38-MAPK

2区 · 医学

Article

作者: D'Ascola, A. ; Scuruchi, M. ; Nastasi, G. ; Prestipino, V. ; Campo, G. M. ; Campo, S. ; Calatroni, A. ; Avenoso, A.

4-mer hyaluronan (HA) oligosaccharides stimulate pro-inflammatory effects in different cell types by interacting with both the toll-like receptor-4 (TLR-4) and -2 (TLR-2). This interaction induces the activation of the transforming growth factor activated kinase-1 (TAK-1) that activates the nuclear factor kappaB (NF-kB) either directly and/or through the activation of p38-mitogen-activated protein kinase (p38-MAPK). This in turn induces the transcription of proinflammatory mediators that prime inflammation. Our aim was to investigate the involvement of TAK-1 and p38-MAPK in 4-mer HA oligosaccharide-induced inflammatory response in mouse synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and from mice subjected to collagen-induced arthritis (CIA). Treatment of NSF and rheumatoid arthritis synovial fibroblasts (RASF) with 4-mer HA showed a marked up-regulation of TLR-4, TLR-2, TAK-1 and p38-MAPK mRNA expression and of the related proteins, as well as NF-kB activation. High levels were also detected of TNF-α, IL- 1β, MMP-13 and iNOS. Treatment of NSF and RASF, previously stimulated with 4-mer HA oligosaccharides, with TAK- 1 and/or p38-MAPK specific inhibitors significantly reduced all the parameters, although the inhibitory effect of p38- MAPK was less effective than that of TAK-1. The addition of CD44 antibody to both NSF and RASF showed that CD44 was not involved in 4-mer HA-induced inflammation.

2007-04-01·EXPERIMENTAL HEMATOLOGY

The role of Wilms' tumor gene peptide-specific cytotoxic T lymphocytes in immunologic selection of a paroxysmal nocturnal hemoglobinuria clone.

Article

作者: Shichishima, Tsutomu ; Maruyama, Yukio ; Akutsu, Kazuko ; Ikeda, Kazuhiko ; Nakamura-Shichishima, Akiko ; Noji, Hideyoshi ; Osumi, Kazuoki ; Yasukawa, Masaki

OBJECTIVE:

To clarify an expansion mechanism of a paroxysmal nocturnal hemoglobinuria (PNH) clone with the Wilms' tumor gene (WT1).

MATERIALS AND METHODS:

In PNH patients with the HLA-A*2402 allele, frequencies of peripheral blood (PB) WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells and WT1 peptide-stimulated interferon-gamma-producing mononuclear cells (MNCs), cytotoxicity of WT1 peptide-specific and HLA-A*2402-restricted cytotoxic T lymphocyte (CTL) clone (TAK-1) cells on bone marrow (BM) MNCs, and after co-incubation with TAK-1 cells, changes in colony-forming unit granulocyte-macrophage colony formation of CD34+ cells and in CD59 expression in viable CD34+ cells were investigated.

RESULTS:

The frequencies of PB WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells (p < 0.005) and WT1 peptide-stimulated interferon-gamma-producing MNCs (p < 0.02) were significantly higher in 5 PNH patients than 8 healthy volunteers (HV). In 5 PNH patients or 3 HV, TAK-1 cells significantly killed BMMNCs and suppressed colony formations of CD34+CD59+ and/or CD34+CD59- cells in the absence and presence of a WT1 peptide or only in the presence of the peptide, respectively, in an HLA-restricted manner. After co-incubation with TAK-1 cells, reduction rates of colony formation of CD34+CD59- cells were significantly less than those of CD34+CD59+ cells in 5 PNH patients (p < 0.002) and proportions of viable CD34+CD59- cells from 5 PNH patients significantly increased in the absence (p < 0.01) and presence (p < 0.01) of a WT1 peptide in an HLA-restricted manner.

CONCLUSION:

WT1 peptide-specific and HLA-restricted CTLs may play an important role in expansion of a PNH clone during immunologic selection and/or in the occurrence of BM failure via interferon-gamma in PNH.

100 项与 TAK1 相关的药物交易

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