Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.

In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the −51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a −51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the −51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the −51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.

WC, Doi T. RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins. J Thromb Haemost 2013; 11: 1742-50.Summary. Background: Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runtrelated transcription factor 1 (RUNX1) of FPD patients. Objectives: To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. Methods: We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFb), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. Results: We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFb, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFb, and FLI-1. Conclusions: RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

The thermal conductivities of resins can be improved by introducing a high-order structure having microscopic anisotropy while maintaining their macroscopic isotropy. We studied four kinds of diepoxy mnnomers with a biphenyl group or two phenyl benzoate groups as mesogens, and cured them thermally with an aromatic diamine curing agent. Their thermal conductivities were up to 5 times higher than those of conventional epoxy resins, because the molecular groups, mesogens form highly ordered crystal-like structures which suppress phonon scattering. We confirmed the existence of crystal-like structures in the epoxy resins directly using transmission electron microscope (TEM) observation. We also observed mezoscopic structures in the resins using an atomic force microscope (AFM). The results suggest a novel method to improve the thermal conductivities of the insulating resins themselves by controlling their highorder structures. Furthermore, the laminates were prepared with the high thermal conductive epoxy resin containing a biphenyl group and ceramic fillers as a part of a feasibility study. Thermal conductivities more than 10 times higher than ordinary epoxy resin laminates were obtained for test pieces of the laminates. 0-7803-85845/04/$20.00 02004 IEEE

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