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Nakayama Y.,Osaka National Research Institute
Accounts of Chemical Research | Year: 2012

Although gene therapy offers an attractive strategy for treating inherited disorders, current techniques using viral and nonviral delivery systems have not yielded many successful results in clinical trials. Viral vectors such as retroviruses, lentiviruses, and adenoviruses deliver genes efficiently; however, the possibility of negative outcomes from viral transformation cannot be completely ruled out. In contrast, various types of nonviral vectors are attracting considerable attention because they are easier to handle and induce weak immune responses. Cationic polymers, such as polyethylenimine (PEI) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm), can generate nanoparticles through the formation of polyion complexes, "polyplexes" with DNA. These nonviral systems offer many advantages over viral systems. The primary obstacle to implementing these cationic polymers in an effective gene therapy remains their comparatively inefficient gene transfection in vivo.We describe four strategies for the development of hyperbranched star vectors (SVs) for enhancing DNA or siRNA delivery. The molecular design was performed by living radical polymerization in which the chain length can be controlled by photoirradiation and solution conditions, including concentrations of the monomer or iniferter (a molecule that serves as a combination of initiator, transfer agent, and terminator). The branch composition is controlled by the types of monomers that are added stepwise. In our first strategy, we prepared a series of only cationic PDMAPAAm-based SVs with no branches or 3, 4, or 6 branching numbers. These SVs could form polyion complexes (polyplexes) by mixing with DNA only in aqueous solution. The relative gene expression activity of the delivered DNA increased according to the degree of branching. In addition, increasing the molecular weight of SVs and narrowing their polydispersity index (PDI) improved their activity. For targeting DNA delivery to the specific cells, we modified the SV with ligands. Interestingly, the SV could adsorb the RGD peptide, making gene transfer possible in endothelial cells which are usually refractory to such treatments. The peptide was added to the polyplex solution without covalent derivatization to the SV. The introduction of additional branching by cross-linking using iniferter-induced coupling reactions further improved gene transfection activity. After block copolymerization of PDMAPAAm-based SVs with a nonionic monomer (DMAAm), the blocked SVs (BSVs) produced polyplexes with DNA that had excellent colloidal stability for 1 month, leading to efficient in vitro and in vivo gene delivery. Moreover, BSVs served as carriers for siRNA delivery. BSVs enhanced siRNA-mediated gene silencing in mouse liver and lung. As an alternative approach, we developed a novel gene transfection method in which the polyplexes were kept in contact with their deposition surface by thermoresponsive blocking of the SV. This strategy was more effective than reverse transfection and the conventional transfection methods in solution. © 2012 American Chemical Society. Source

Regenerative medicine using human stem cells is one of the newest and most promising fields for treating various intractable diseases and damaged organs. For clinical applications, choosing which human stem cells to use, i.e. according to tissue of origin and progenitor type, is a critical issue. Neural stem/progenitor cells (NSPCs) hold promise for treating various neurological diseases. We have shown that the transporter protein ABCB1 is predominantly expressed in immature human fetal NSPCs, and thus could be used as a phenotypic marker to investigate and monitor NSPCs in culture. We describe our proposed model for the in vitro proliferative process of aggregated human NSPCs and show that neurosphere enlargement and NSPC proliferation are mutually reinforcing. We have established that human neurospheres contain a heterogeneous cell population, knowledge that will contribute to the development of human neurospheres with desirable characteristics for clinical applications. Furthermore, decidua-derived mesenchymal cells (DMCs), which we isolated from human placenta, have unique properties as mesenchymal stem cells. They also generate a pericellular matrix (PCM-DM) that supports the growth and pluripotency of human embryonic stem cells and induced pluripotent stem cells (hiPS) cells. The newly developed re-programming techniques for generating hiPS cells should greatly contribute to cell therapies using human pluripotent stem cells, including those derived from DMCs. Our DMC-derived hiPS cells are a promising candidate source of allogeneic hiPS cells for clinical applications. We hope our findings will contribute to the development of cell-culture systems for generating human allogeneic stem cells for clinical use in regenerative medicine. Source

Takeda S.,Osaka National Research Institute | Takeya H.,Tottori University | Iwanaga S.,Chemo Sero Therapeutic Research Institute
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2012

Metalloproteinases are among the most abundant toxins in many Viperidae venoms. Snake venom metalloproteinases (SVMPs) are the primary factors responsible for hemorrhage and may also interfere with the hemostatic system, thus facilitating loss of blood from the vasculature of the prey. SVMPs are phylogenetically most closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif) family of proteins and, together with them, constitute the M12B clan of metalloendopeptidases. Large SVMPs, referred to as the P-III class of SVMPs, have a modular architecture with multiple non-catalytic domains. The P-III SVMPs are characterized by higher hemorrhagic and more diverse biological activities than the P-I class of SVMPs, which only have a catalytic domain. Recent crystallographic studies of P-III SVMPs and their mammalian counterparts shed new light on structure-function properties of this class of enzymes. The present review will highlight these structures, particularly the non-catalytic ancillary domains of P-III SVMPs and ADAMs that may target the enzymes to specific substrates. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. © 2011 Elsevier B.V. All rights reserved. Source

Shimizu S.,University of Toyama | Kaiya H.,Osaka National Research Institute | Matsuda K.,University of Toyama
Peptides | Year: 2014

Ghrelin is a potent orexigenic peptide implicated in appetite regulation in rodents. However, except for teleost fish, the involvement of ghrelin in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and prometamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in growing larvae. In the present study, we examined the effect of intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic bullfrog ghrelin (n-octanoylated 28-amino acid form) on food intake in larvae at the prometamorphic stages. Cumulative food intake was significantly increased by IP (8 and 16 pmol/g body weight (BW)) or ICV (0.5 and 1 pmol/g BW) administration of ghrelin during a 15-min observation period. The orexigenic action of ghrelin at 8 pmol/g BW (IP) or at 0.5 pmol/g BW (ICV) was blocked by treatment with a growth hormone secretagogue-receptor antagonist, [D-Lys3]GHRP-6 at 80 pmol/g BW (IP) or at 5 pmol/g BW (ICV). We then investigated the effect of feeding status on expression levels of the ghrelin transcript in the hypothalamus and gastrointestinal tract. Ghrelin mRNA levels in both were decreased 15 and 60 min after feeding. These results indicate that ghrelin acts as an orexigenic factor in bullfrog larvae. © 2013 Elsevier B.V. All rights reserved. Source

Kojima M.,Kurume University | Kangawa K.,Osaka National Research Institute
Endocrine Development | Year: 2013

Since its discovery 12 years ago, intensive research has been performed on ghrelin. The significance of ghrelin as a growth hormone-releasing hormone, appetite regulator, energy conservator and sympathetic nerve suppressor has now been well established. In this short essay, we summarize the history of the discovery of ghrelin. Copyright © 2013 S. Karger AG, Basel. Source

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