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Akitsu Masuda,Jian Xu,Kosuke Minamihata,Genki Kagawa,Yusei Hamada,Yoshiki Morifuji,Takumi Yano,Masato Hino,Daisuke Morokuma,Noriko Karasaki,Hiroaki Mon,Noriho Kamiya,Takahiro Kusakabe,이재만 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.2
As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/ larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.
The Effects of Components of Grazing System on Welfare of Fattening Pigs
Tozawa, Akitsu,Tanaka, Shigefumi,Sato, Shusuke Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.3
The objective of this study was to clarify the most effective component of grazing for improving welfare of fattening pigs. This study compared welfare indicators of 20 fattening pigs aged 100 to 124 days (the prior period) and 138 to 164 days (the latter period) in an indoor housing system (IS), an outdoor pasturing system (OP), a concrete floor paddock system (CF), a concrete floor paddock system with fresh grass (FG), or a soil floor paddock system (SF). The last three treatments include important components of a grazing system: extra space, grass feed, and soil floor. Behavior, wounds on the body, and performances, measured as average daily gain (ADG) and feed conversion ratio, were observed. CF pigs behaved similarly to IS pigs. FG pigs showed higher levels of foraging, chewing and activity. SF pigs engaged in higher levels of foraging, exploring, activity, and rooting, and showed a similar amount of playing behavior as OP pigs. ADG was the same in all treatments at the prior period, and increased in the order FG, IS, CF, SF, and OP at the latter. The behaviors and performance of SF pigs resembled those of OP which seemed to indicate a consistently higher standard of welfare than the other treatments. In conclusion, the existence of a soil floor is the most important component of a pasture for improving the welfare of pigs.
Moon, Dohyun,Tanaka, Shinnosuke,Akitsu, Takashiro,Choi, Jong-Ha Elsevier 2018 Journal of molecular structure Vol.1154 No.-
<P><B>Abstract</B></P> <P>Two new copper (II) complexes, [Cu(<I>N</I>-Metn)<SUB>2</SUB>Cl]BF<SUB>4</SUB> (<B>1</B>) and [Cu(Me<SUB>2</SUB>tn)<SUB>2</SUB>(N<SUB>3</SUB>)]N<SUB>3</SUB> (<B>2</B>) (<I>N</I>-Metn = <I>N</I>-methyl-1,3-propanediamine; Me<SUB>2</SUB>tn = 2,2-dimethyl-1,3-propanediamine), have been prepared, fully characterized, and their structures established by X-ray single-crystal analysis from synchrotron diffraction data. For these complexes, the Cu(II) ions are five-coordinate in an axially elongated square-pyramidal environment, with the four amine N atoms at the equatorial positions and the Cl atom (<B>1</B>) or N atom of one azide (<B>2</B>) at an apical site. The CuN bond lengths for amine N atoms in the complex <B>1</B> (2.0198(13)–2.0735(12) A°) and complex <B>2</B> (2.0239(9)–2.0489(9) Å) are typical, but the axial ligands are coordinated with a CuCl bond length of 2.5962 (7) Å for complex <B>1</B>, and the CuN (azido) bond length is 2.1990 (10) Å for complex <B>2</B>, adopting square-planar geometry around the Cu(II) with four N atoms from two <I>N</I>-Metn or Me<SUB>2</SUB>tn ligands. The crystals are stabilized by a three-dimensional network of intermolecular hydrogen bonds that are formed between the primary and secondary amine groups of the <I>N</I>-Metn ligands, the Cl ligands, and the F atoms of BF<SUB>4</SUB> <SUP>−</SUP> anion in <B>1</B>, and by the primary amine groups of the Me<SUB>2</SUB>tn ligands and the N atoms of the azido ligand and azide ion in <B>2</B>. Hirshfeld surface analysis with 2D fingerprint plots revealed that the H⋯H, F⋯H, Cl⋯H contacts in <B>1</B> and the H⋯H and N⋯H contacts in <B>2</B> are the main intermolecular interactions. The electronic absorption and IR spectral properties are also discussed.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Syntheses of two new Cu(II) complexes. </LI> <LI> Two Cu(II) complexes are structurally characterized through single crystal X-ray diffraction. </LI> <LI> Spectroscopic and magnetic properties including Hirshfeld surface analysis are also described. </LI> </UL> </P>
Akihiro Morio,Jian Xu,Akitsu Masuda,Yurie Kinoshita,Masato Hino,Daisuke Morokuma,Hatsumi M. Goda,Nozomu Okino,Makoto Ito,Hiroaki Mon,Ryosuke Fujita,Takahiro Kusakabe,이재만 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2
The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial Oglycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.
Kakino Kohei,Masuda Akitsu,Hino Masato,Ebihara Takeru,Xu Jian,Mon Hiroaki,Fujita Ryosuke,Fujii Tsuguru,Kusakabe Takahiro,Lee Jae Man 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.3
Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkwormbaculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.
( Daisuke Morokuma ),( Masato Hino ),( Miho Tsuchioka ),( Akitsu Masuda ),( Hiroaki Mon ),( Kazuhito Fujiyama ),( Hiroyuki Kajiura ),( Takahiro Kusakabe ),( Jae Man Lee ) 한국잠사학회 2018 International Journal of Industrial Entomology Vol.36 No.1
N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.