記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biochemical Engineering Journal  

Bile acid (BA) micelle-disrupting peptides are bioactive peptides (BPs) that suppress intestinal cholesterol adsorption and ameliorate blood cholesterol levels. To facilitate their application in the food industry, an effective and straightforward technology for BP separation and enrichment of BPs is required. For this purpose, we assessed the potential of heat-treated (HT) porous silica gels with an enhanced capacity for peptide adsorption/desorption (AD). Given the key characteristics of these peptides involving basic amino acid (AA) residues, we applied casein hydrolysate to HT silica gels for adsorption at neutral pH, where the surface yielded anionic silanolates through deprotonation. Subsequently, desorption was conducted at pH 2, at which point the surface returned to its neutral state. We confirmed a 335-fold increase in the BA micelle-disrupting activity of the resulting hydrolysates. LC-MS/MS analysis identified 783 peptides, confirming that the peptides containing cationic AA residues were selectively enriched, as expected. Moreover, a dose-response of randomly selected 27 peptides with various enrichment levels revealed the presence of multiple active peptides in the enriched groups. These results indicate that our method is effective for the separation and enrichment of BA micelle-disrupting peptides and holds promise for BP production.

DOI： 10.1016/j.bej.2024.109283

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biochemical and Biophysical Research Communications  

Extracellular vesicles (EVs) released into the blood during exercise mediate its whole-body health effects. The differentiation of EVs released by skeletal muscle cells in vivo from those released by other cells is challenging, therefore, it is unclear whether exercise increases the number of EVs secreted by skeletal muscle cells. In this study, we investigated whether exercise affects the quantity of EVs released from skeletal muscle cells using in vitro exercise models. C2C12 myotubes were cultured on a gel layer with 1 or 30 Hz electrical pulse stimulation (EPS) to induce contractions as an artificial simulating exercise. We found that tetanic contraction induced by 30 Hz EPS increased the number of secreted EVs. MicroRNA (miRNA)-seq analysis revealed that 30 Hz EPS altered the miRNA in the secreted EVs. Furthermore, expression analysis of genes related to the biogenesis and transport of EVs revealed that the expression of ALG-2 interacting protein X (Alix) was increased in response to 30 Hz EPS, and the peak value of intracellular Ca2+ in myotubes at 30 Hz EPS was higher than that at 1 Hz, indicating that the increase in intracellular Ca2+ concentration may be related to the increased secretion of EVs in response to 30 Hz EPS.

DOI： 10.1016/j.bbrc.2023.06.054

Web of Science

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cells  

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

DOI： 10.3390/cells11233760

Web of Science

Scopus

PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：公益社団法人 日本生物工学会  

<p>プロテアーゼの切断点推定方法を確立するため，4残基1990種のペプチド配列をデザインし，末端蛍光ラベルしたライブラリーを合成し，酵素分解を行い，そのデータを使ってRandom Forest（RF）で切断点推定モデルを構築した．1990配列は切断点になるジペプチドをすべて網羅している．RFモデルに利用する変数としてアミノ酸やジペプチドの存在だけでなく，ペプチドの特性を示すため位置変数（Positioning parameter, PP），および包括変数（Global parameter, GP）も考案した全532変数を用いた．プロテアーゼとして，生化学用試薬グレードのトリプシンを使用し，pH 8で切断し切断率をヒストグラムで評価した結果，予想通りアルギニン残基（R），リジン残基（K）を含むペプチド配列は切断しやすいことがわかった．構築したpH 8-RFモデルの推定精度は77 ％にとどまった．RFモデルで重要度の高い10変数として，Kの存在と9個のGPが選ばれ，GPの中で，R，Kが特徴的に大きい値をとる等電点に関する4変数と極性に関する変数1個が含まれた．α-lactalbuminの切断点を予測し，実際に酵素分解し切断ペプチドをLC-MS/MSで解析したところ，42種類のペプチドが同定された．RFモデルで予測した19か所のうち14か所が一致した．69Y-70G間や50F-51H間で切断されたペプチド断片が多く，いずれもトリプシンではなく混入するキモトリプシンによる切断が強く示唆された．この部位についても構築したモデルで切断が予測できた．さらに，pH 5でペプチド切断を行い，トリプシンの加水分解に及ぼすpHの影響を調べた．pH 5のモデルの推定精度は残念ながら59 ％と低かった．構築したpH 5-RFモデルでは重要度の高い10変数としてpH 8モデルと同じ等電点に関する4個のGPが含まれた．シグナル配列以降は，pH 8での結果とほぼ同数の20か所となった．同様にpH 5でα-lactalbuminをトリプシンにより加水分解し64種の断片を同定した．検出断片が多かった48Tから77Kまでの間で推定予測部位と比較したところ，新規に増加した多くの部位で構築したモデルでも切断が予測できることに加え，pHを変えることで生じる切断特異性の変化をある程度捉えることができた．今回の成果は，1990種のペプチドライブラリーと532変数で構築したRFモデルが，プロテアーゼ切断点推定に効果的であることを明らかにした．</p>

DOI： 10.34565/seibutsukogaku.100.10_528

DOI： 10.34565/seibutsukogaku.100.10_528

Scopus

CiNii Research

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biotechnology and Bioengineering  

Skeletal muscle atrophy is characterized by decreases in protein content, myofiber diameter, and contractile force generation. As muscle atrophy worsens the quality of life, the development of anti-atrophic substances is desirable. In this study, we aimed to demonstrate a screening process for anti-atrophic peptides using photo-cleavable peptide array technology and human contractile atrophic muscle models. We developed a 96-well system and established a screening process with less variability. Dexamethasone-induced human atrophic tissue was constructed in the system. Eight peptides were selected from the literature and used for the screening of peptides for preventing the decrease of the contractile forces of tissues. The peptide QIGFIW, which showed preventive activity, was selected as the seed sequence. As a result of amino acid substitution, we obtained QIGFIQ as a peptide with higher anti-atrophic activity. These results indicate that the combinatorial use of the photo-cleavable peptide array technology and 96-well screening system could comprise a powerful approach to obtaining anti-atrophic peptides, and suggest that the 96-well screening system and atrophic model represent a practical and powerful tool for the development of drugs/functional food ingredients.

DOI： 10.1002/bit.28125

Web of Science

Scopus

PubMed

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/bit.28125