Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：Springer US  

Pas2r12 is comprised of a repeat of the penetration-accelerating sequence (Pas) (Pas2: FFLIG-FFLIG) and D-form dodeca-arginine (r12), a cell-penetrating peptide. Pas2r12 significantly enhances cytosolic delivery of cargo proteins, including enhanced green fluorescent protein and immunoglobulin G. Simply incubating Pas2r12 with cargo leads to their cytosolic tranlsocation. Cytosolic delivery of cargo by Pas2r12 involves caveolae-mediated endocytosis. In this chapter, we describe methods of cytosolic delivery of cargo using Pas2r12 and provide methods for investigating the cellular uptake pathway of cargo by Pas2r12.

DOI： 10.1007/978-1-0716-1752-6_18

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Language：Japanese   Publisher：(株)北隆館  

我が国は世界でも最高位に位置する長寿国となり、日本人の平均寿命・健康寿命は共に世界第1位となっているが、平均寿命と健康寿命の間には乖離があり、晩年の約10年間を「自立した生活ではない期間」として過ごしていることとなる。高齢者の健康寿命の障害となるものフレイル・ロコモ・サルコペニアを予防・改善することが健康寿命を延伸していくために大切である。フレイル・ロコモ・サルコペニアの共通項目である筋力・筋肉量低下の原因となる栄養障害の1つに、亜鉛欠乏による味覚障害があり、亜鉛補給による味覚障害の改善は食欲・食事摂取量を増進しフレイル・ロコモ・サルコペニアの予防・改善につながる可能性がある。(著者抄録)

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Authorship：Lead author,　Corresponding author  

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：The Society of Analytical Bio-Science  

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Language：Japanese   Publisher：(株)ジェフコーポレーション  

市販の油淋鶏弁当の主食:副食の割合を10g:3g、10g:6g、10g:9gに調整し、この順序で一口に食し主観的味覚を点数化した。20〜25歳の健常な日本人女性12名とスリランカ人女性4名を対象とした。副食が増量していくに従い日本人は酸味・渋味・旨味の評価が高くなっていったが、スリランカ人では塩味・甘味・苦味の評価が高くなった。同様の3パターンの食事を味覚センサーで分析した。その結果、白飯をコントロールとした場合、副食の増量に従い酸味・旨味が高くなり、炒飯をコントロールとした場合は酸味が強く影響され、旨味や渋味は副食を6gより増加させてもほとんど変化しなかった。コントロールとして白飯を用いた場合と炒飯を用いた場合とでは味覚のパターンが異なり、日本人・スリランカ人各々の主観的味覚評価のいずれともパターンが一致しなかった。

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Language：Japanese   Publisher：(株)ジェフコーポレーション  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

DOI： 10.1021/acs.biomac.8b01299

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Language：English   Publishing type：Research paper (scientific journal)  

Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21, which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs.

DOI： 10.1016/j.bbrc.2019.01.025

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：日本外科代謝栄養学会  

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Language：Japanese   Publisher：(株)ジェフコーポレーション  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Poly(ADP-ribose) polymerase 1 (PARP-1), which catalyzes poly(ADP-ribosyl)ation of proteins by using NAD+ as a substrate, plays a key role in several nuclear events, including DNA repair, replication, and transcription. Recently, PARP-1 was reported to participate in the somatic cell reprogramming process. Previously, we revealed a role for PARP-1 in the induction of neural apoptosis in a cellular model of cerebral ischemia and suggested the possible use of PARP inhibitors as a new therapeutic intervention. In the present study, we examined the effects of PARP inhibitors on neural stem/progenitor cells (NSPCs) of the mouse brain.
Results: PARP-1 was more abundant and demonstrated higher activity in NSPCs than in mouse embryonic fibroblasts. Treatment with PARP inhibitors suppressed the formation of neurospheres by NSPCs through the suppression of cell cycle progression and the induction of apoptosis. In order to identify the genes responsible for these effects, we investigated gene expression profiles by microarray analyses and found that several genes in the p53 signaling pathway were upregulated, including Cdkn1a, which is critical for cell cycle control, and Fas, Pidd, Pmaip1, and Bbc3, which are principal factors in the apoptosis pathway. Inhibition of poly(ADP-ribosyl) ation increased the levels of p53 protein, but not p53 mRNA, and enhanced the phosphorylation of p53 at Ser18. Experiments with specific inhibitors and also shRNA demonstrated that PARP-1, but not PARP-2, has a role in the regulation of p53. The effects of PARP inhibitors on NSPCs were not observed in Trp53(-/-) NSPCs, suggesting a key role for p53 in these events.
Conclusions: On the basis of the finding that PARP inhibitors facilitated the p53 signaling pathway, we propose that poly(ADP-ribosyl) ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation.

DOI： 10.1186/s12868-016-0333-0

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Language：Japanese   Publisher：一般社団法人 日本不整脈心電学会  

交感神経興奮は器質的心疾患症例の心室不整脈に対する催不整脈効果を有し，迷走神経興奮は抗不整脈作用を示すと考えられる．自律神経興奮はその強度のみならず，交感神経興奮と迷走神経興奮のバランス，変動のダイナミクスも不整脈発症に影響を及ぼす．日常診療では，器質的心疾患を有する心室不整脈に，心機能と不整脈の両面からβ遮断薬が用いられるとともに，β遮断薬作用を併せもつIII群抗不整脈薬が処方されている．繰り返し生じる心室頻拍によりelectrical stormに陥った場合，自律神経は重要な治療介入ポイントとなる．初期治療では鎮痛・鎮静・麻酔とともにβ遮断薬が用いられるが，重症例では短期作用型β遮断薬静注，さらには心臓交感神経のブロック・切除，腎動脈カテーテルアブレーション，硬膜外ブロックなどの非薬物治療が用いられる場合があり，交感神経興奮抑制を介した一定の治療効果が期待できる．

DOI： 10.5105/jse.36.31

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.

DOI： 10.1038/mt.2009.192

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

There are a growing number of reports showing the usefulness of cell-penetrating peptides (CPPs) including oligoarginines for intracellular delivery of macromolecules. Although the covalent attachment of the CPP segments to the cargo molecules is usually required to ensure effective delivery, conventional methods of conjugation need several manipulation steps that are often time-consuming and laborious. Here, we report a novel approach to allow easy modification of cargo molecules with oligoarginine CPPs. The key feature is the employment of oligoarginines (R8 and R12) equipped with a sulfosuccinimidylsuberyl moiety (BS(3)-R8 and -R12). One-pot modification is achieved simply by mixing BS(3)-R8 and -R12 with cargos in an aqueous buffer. The usefulness of this approach was exemplified through the conjugate formation with Fab fragments of immunoglobulin G, amino-functionalized poly(ethylene glycol)s (amino-PEGs), and amino quantum dots (amino-QDs), yielding an efficient cellular uptake.

DOI： 10.1021/bc800327f

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Language：English   Publishing type：Research paper (international conference proceedings)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

As the versatility and use of CPPs (cell-penetrating peptides) as intracellular delivery vectors have been widely accepted, the cellular uptake mechanisms that enable their efficient internalization have become the subject of much interest. Arginine-rich peptides, including HIV-1 Tatp (transactivator of transcription peptide), are regarded as a representative class of UPS. Evidence suggests that macropinocytosis plays a crucial role in the cellular uptake of these peptides. we have recently shown that treatment of cells with arginine-rich peptides induces activation of Rac protein leading to F-actin (filamentous actin) organization and macropinocytosis. We have also shown that depletion of membrane-associated proteoglycans results in the failure of this signalling pathway, suggesting that membrane-associated proteoglycans may act as a potential receptor for the induction of macropinocytic uptake of arginine-rich peptides. However, when the macropinocytic pathway is inhibited at a low temperature or by cholesterol depletion, these peptides can be internalized by alternative mechanisms, one of which appears to be direct translocation of the peptides through the plasma membrane. This review summarizes the current theories on both endocytic and non-endocytic aspects of internalization of arginine-rich peptides.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.

DOI： 10.1021/bi0612824

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

Successful intracellular delivery of various bioactive molecules has been reported using cell permeating peptides (CPPs) as delivery vectors. To determine the effects of CPPs on the cellular uptake of immunoglobulin Fab fragment, conjugates of a radio-iodinated Fab fragment with CPPs (CPP-I-125-Fab) derived from HIV-1 TAT, HIV-1 REV, and Antennapedia (ANP) were prepared. These vectors are rich in basic amino acids, and their strong adsorption on cell surfaces often results in overestimation of internalized peptides. Cell wash with an acidic buffer (0.2M glycine-0.15M NaCl, pH 3.0) was thus employed in this study to remove cell-surface adsorbed CPP-I-125-Fab conjugates. This procedure enabled clearer understanding of the methods of internalization of CPP-I-125-Fab conjugates. The kinetics of internalization of REV-I-125-Fab conjugate was rapid, and a considerable fraction of REV-I-125-Fab was taken up by HeLa cells as early as 5 min after administration. It was also shown that cellular uptake of these conjugates was significantly inhibited in the presence of endocytosis/macropinocytosis inhibitors, in the order REV-I-125-Fab > TAT-I-125-Fab >= ANP-I-125-Fab; this order was the same as for effectiveness of intracellular delivery. Simultaneous cell washing with phosphate-buffered saline (PBS) and this acidic buffer effectively separated the internalized conjugates from the cell-surface-adsorbed ones, and considerable differences were observed in these amounts dependent on the employed CPPs. (c) 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 98-107, 2007.

DOI： 10.1002/bip.20689

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Authorship：Lead author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Publishing type：Research paper (international conference proceedings)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosotic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal. primary cultured neurons. Enhancement of bioactivity on the administration of an apoptosis-inducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.

DOI： 10.1021/cb600127m

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Arginine-rich peptide-mediated protein delivery into living cells is a novel technology for controlling cell functions with therapeutic potential. In this report, a novel approach for the intracellular delivery of histidine-tagged proteins was introduced where a Ni(II) chelate of octaarginine peptide bearing nitrilotriacetic acid [R8-NTA-Ni(II)] was used as a membrane-permeable carrier molecule. Significant internalization of histidine-tagged enhanced green fluorescent protein (EGFP) into HeLa cells was observed by confocal microscopic observation in the presence of R8-NTA-Ni(II). Nuclear condensation characteristic in apoptotic cell death was also induced in the cells treated with a histidine-tagged apoptosis-inducing peptide [pro-apoptotic domain peptide (PAD)], indicating that the cargo molecules really went through the membrane to reach the cytosol. The apoptosis-inducing activity of the peptide thus delivered was compared with that of the PAD peptide covalently connected with the octaarginine peptide.

DOI： 10.1021/bc034181g

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Escherichia coli spr (suppressor of prc) mutants and nlpI mutants show thermosensitive growth. The thermosensitivity of the spr mutants was suppressed by the nlpI mutations. Expression of the fusion genes encoding hexa-histidine-tagged NlpI (NIpI-His) and purification of the tagged Nlp1 showed that NlpI-His bound with Pre protease and IbpB chaperone. NlpI-His with the amino acid substitution of G103D did not bind with either of these proteins, while NlpI-His variants (NlpI-284-His, NlpI-Q283-His, and NlpI-G282-His) lacking 10 to 12 residues from the carboxy terminus bound with both proteins. The tagged NlpI lacking 11 amino acid residues from the carboxy terminus was processed by Prc, but that lacking 12 residues was not. The thermosensitivity of the nlpI mutant was corrected by the production of the former NlpI variant, but not by production of the latter. Expression of the truncated NlpI that lacked 10 or 11 residues from the carboxy terminus corrected the thermosensitivity of the prc nlpI double mutant, while expression of the full-length Nlp1 did not. Thus, it was suggested that NlpI was activated by Pre protease processing.

DOI： 10.1093/jb/mvh022

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