Higashihiroshima, Japan
Higashihiroshima, Japan

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Urano S.,Niigata University | Kaneko C.,Niigata University | Nei T.,Niigata University | Motoi N.,Niigata University | And 6 more authors.
Journal of Immunological Methods | Year: 2010

The aim of the project is to develop a novel method estimating granulocyte-macrophage colony-stimulating factor (GM-CSF) neutralizing capacity with high-throughput and good reproducibility. For that purpose, we designed a cell-free receptor binding assay consisting of a solid-phase recombinant soluble GM-CSF receptor α (GMRα) and a biotinylated GM-CSF (bGM-CSF). Using this system, competitive inhibition of bGM-CSF binding to soluble GM-CSF receptor α (sGMRα) by GM-CSF autoantibody or IgG fractions from the sera of patients with pulmonary alveolar proteinosis was examined, resulting in excellent reproducibility. Binding inhibition was correlated with growth inhibition of TF-1 cells, a GM-CSF dependent cell line. These results suggest that our cell-free system can be applied to estimate the neutralizing capacity of GM-CSF autoantibodies ex vivo. © 2010 Elsevier B.V.

Kitajima T.,RIKEN | Obuse S.,RIKEN | Adachi T.,Neosilk Co. | Tomita M.,Neosilk Co. | Ito Y.,RIKEN
Biotechnology and Bioengineering | Year: 2011

The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering. © 2011 Wiley Periodicals, Inc.

Adachi T.,Neosilk Co. | Wang X.,Neosilk Co. | Murata T.,Neosilk Co. | Obara M.,Hiroshima University | And 4 more authors.
Biotechnology and Bioengineering | Year: 2010

We generated transgenic silkworms that synthesized human type I collagen α1 chain [α1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant a1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the α1(I) chain content to 8.0% (4.24 mg per cocoon). The a1(I) chain was extracted and purified from the cocoons using a very simple method. The α1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the α1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the α1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the α1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human a1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture. © 2010 Wiley Periodicals, Inc.

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