Artificial Extracellular Matrix Proteins Containing Phenylalanine Analogues Biosynthesized in Bacteria Using T7 Expression System and the PEGylation

In vivo incorporation of phenylalanine (Phe) analogues into an artificial extracellular matrix protein (aECM-CS5-ELF) was accomplished using a bacterial expression host that harbors the mutant phenylalanyl-tRNA synthetase (PheRS) with an enlarged binding pocket. Although the Ala294Gly/Thr251Gly mutant PheRS (PheRS**) under the control of T5 promoter allows incorporation of some Phe analogues into a protein, the T5 system is not suitable for material science studies because the amount of materials produced is not sufficient due to the moderate strength of the T5 promoter. This limitation can be overcome by using a pair of T7 promoter and T7 RNA polymerase instead. In the T7 expression system, it is difficult, however, to achieve a high incorporation level of Phe analogues, due to competition of Phe analogues for incorporation with the residual Phe that is required for synthesis of active T7 RNA polymerase. In this study, we prepared the PheRS** under T7 promoter and optimized culture condition to improve both the incorporation level of recombinant aECM protein and the incorporation level of Phe analogues. Incorporation and expression levels tend to increase in the case of p-azidophenylalanine, p-iodophenylalanine, and p-acetylphenylalanine. We evaluated the lower critical transition temperature, which is dependent on the incorporation ratio and the turbidity decreased when the incorporation level increased. Circular dichromism measurement indicated that this tendency is based on conformational change from random coil to β-turn structure. We demonstrated that polyethylene glycol (PEG) can be conjugated at reaction site of Phe analogues incorporated. We also demonstrated that the increased hydrophilicity of elastin-like sequences in the aECM-CS5-ELF made by PEG conjugation could suppress nonspecific adhesion of human umbilical vein endothelial cells (HUVEC)

Role of the tumor suppressor APC and the guanine-nucleotide exchange factor Asef in colorectal tumorigenesis

学位の種別:課程博士University of Tokyo(東京大学

Incidence and Risk Factors for Infections Requiring Hospitalization, Including Pneumocystis Pneumonia, in Japanese Patients with Rheumatoid Arthritis

Objective. Rheumatoid arthritis (RA) may be complicated by different infections, but risk factors for these are not fully elucidated. Here, we assessed the incidence of and risk factors for infections requiring hospitalization (IRH) including pneumocystis pneumonia (PCP) in patients with RA. Methods. We retrospectively surveyed all RA patients treated at our hospital from 2009 to 2013, for whom data were available on demographic features, medications, comorbidities, and severity of RA. Multivariate logistic regression analysis was applied to calculate adjusted odds ratios (ORs) for factors associated with the occurrence of IRH. Results. In a total of 9210 patient-years (2688 patients), there were 373 IRH (3.7/100 patient-years). Respiratory tract infections were most frequent (, and additionally 16 PCP), followed by urinary tract infections (). Significant factors for PCP included higher age (≥70 years; OR 3.5), male sex (6.6), underlying lung disease (3.0), use of corticosteroids (4.8), and use of biologics (5.4). Use of methotrexate (5.7) was positively associated with PCP but negatively with total infections (0.7). Additionally, functional disorders and higher RA disease activity were also related to total infections. Conclusions. Risk factors for infection should be taken into account when deciding treatment for the individual RA patient

The miR-1-NOTCH3-Asef Pathway Is Important for Colorectal Tumor Cell Migration

<div><p>The tumor suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumors. APC stimulates the activity of the Cdc42- and Rac1-specific guanine nucleotide exchange factor Asef and promotes the migration and invasion of colorectal tumor cells. Furthermore, Asef is overexpressed in colorectal tumors and is required for colorectal tumorigenesis. It is also known that NOTCH signaling plays critical roles in colorectal tumorigenesis and fate determination of intestinal progenitor cells. Here we show that NOTCH3 up-regulates Asef expression by activating the Asef promoter in colorectal tumor cells. Moreover, we demonstrate that microRNA-1 (miR-1) is down-regulated in colorectal tumors and that miR-1 has the potential to suppress NOTCH3 expression through direct binding to its 3’-UTR region. These results suggest that the miR-1-NOTCH3-Asef pathway is important for colorectal tumor cell migration and may be a promising molecular target for the treatment of colorectal tumors.</p> </div

The adenomatous polyposis coli-associated exchange factors Asef and Asef2 are required for adenoma formation in ApcMin/+mice

Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)-associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine-nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in ApcMin/+ mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC–Asef/Asef2 complex induces c-Jun amino-terminal kinase-mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatement of colorectal tumours

OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

ヒト角膜上皮分化を規定している新分子を発見－OVOL2は角膜上皮における上皮性恒常維持に関与－. 京都大学プレスリリース. 2016-05-09.In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocally repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition

Involvement of endothelial DLL4 in NOTCH3/Asef-mediated cell migration.

<p>(A) Suppression of DLL4 expression by siRNA. Lysates prepared from HUVECs transfected with an siRNA targeting DLL4 or control siRNA were analysed by immunoblot with antibodies against DLL4. Antibodies against α-tubulin were used as a control. Arrowhead shows DLL4 band; asterisk indicates a non-specific band. (B) HUVECs transfected with an siRNA targeting DLL4 or control siRNA were grown to confluency on polycarbonate filters in the Transwell chambers. DLD-1 cells were subjected to migration assays. (C) DLD-1 cells that had been transfected with Pre-miR-1 were also transfected with either N3ICD or Asef and assayed for migration activity. Results obtained are expressed as the means ±SD of at least three independent experiments. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

NOTCH3/CSL transactivates the Asef promoter.

<p>(A) Schematic representation of the Asef-b promoter region. Predicted CSL-binding sites are indicated by boxes P, the regions corresponding to the primers used for ChIP assays. TSS, Transcription start site. (B) (Left) Schematic diagrams of reporter constructs used for luciferase assays. Fragments of the Asef-b promoter were cloned upstream of the luciferase (Luc) gene. Potential CSL-binding sites are indicated by gray boxes. (Right) Caco-2 cells were transfected with N3ICD or control vector along with reporter constructs containing Asef-b promoter sequences and subjected to luciferase assays. pRL-TK vector was used as an internal control. (C) ChIP assays were performed on DLD-1 cells using anti-CSL antibody or non-specific IgG. The promoter region of <i>Asef-b</i> was enhanced in the immunoprecipitates. The promoter regions of <i>GAPDH</i> and <i>Hes1</i> were used as negative and positive controls, respectively. Results are expressed as the means of the percentage of the input ± SD. *<i>p</i> < 0.05.</p