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1.
Nat Commun ; 12(1): 6623, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34799548

RESUMO

During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.


Assuntos
Regeneração do Cérebro/fisiologia , Hidrogéis/química , Peptídeos/química , Proteínas/química , Adesivos , Animais , Engenharia Biomédica , Lesões Encefálicas/diagnóstico por imagem , Adesão Celular , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/química , Hidrogéis/uso terapêutico , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Nanofibras , Sistema Nervoso , Peptídeos/uso terapêutico , Fator A de Crescimento do Endotélio Vascular
2.
Chemistry ; 27(36): 9197, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34114266

RESUMO

Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.


Assuntos
Hidrogéis , Nanofibras , Adesivos , Peptídeos
3.
Chemistry ; 27(36): 9295-9301, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-33871881

RESUMO

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.


Assuntos
Hidrogéis , Nanofibras , Adesivos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos
4.
ACS Appl Bio Mater ; 3(6): 3605-3611, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-35025230

RESUMO

Cell adhesion is a fundamental biological process involved in a wide range of cellular and biological activity. Integrin-ligand binding is largely responsible for cell adhesion with an extracellular matrix, and the RGD sequence is an epitope in ligand proteins such as fibronectin. The extracellular matrix consists of fibrous proteins with embedded ligands for integrins. Such a biological architecture has been reconstructed for biochemical, pharmaceutical, and biomaterial studies using artificial supramolecular systems to reproduce cell adhesion functionality, and fiber-forming self-assembling peptides containing RGD are one such promising material for this purpose. In this study, using RADA16 as a model fiber-forming peptide, a series of RGD-containing variants have been synthesized by the replacement of one alanine with glycine at different positions, in which all the variants consist of identical amino acid components. The position of the RGD unit influenced the supramolecular self-assembly of the amphiphilic peptide to inhibit ß-sheet formation (A6G) or twist the molecular alignment in ß-sheet-type assemblies (A10G and A14G). Furthermore, A10G and A14G formed assembled nanofibers, which afforded hydrogels with higher viscoelasticities than other RGD-containing variants. In contrast to A10G and A14G, which exhibit substantial cell adhesion functionality, the cell adhesion efficiencies of the other RGD-containing variants were significantly reduced. This suggests that the higher order structure could strongly influence the cell adhesion functionality of RGD-containing supramolecular nanofibers.

5.
Angew Chem Int Ed Engl ; 58(49): 17827-17833, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31544993

RESUMO

Photon upconversion (UC) from near-infrared (NIR) light to visible light has enabled optogenetic manipulations in deep tissues. However, materials for NIR optogenetics have been limited to inorganic UC nanoparticles. Herein, NIR-light-triggered optogenetics using biocompatible, organic TTA-UC hydrogels is reported. To achieve triplet sensitization even in highly viscous hydrogel matrices, a NIR-absorbing complex is covalently linked with energy-pooling acceptor chromophores, which significantly elongates the donor triplet lifetime. The donor and acceptor are solubilized in hydrogels formed from biocompatible Pluronic F127 micelles, and heat treatment endows the excited triplets in the hydrogel with remarkable oxygen tolerance. Combined with photoactivatable Cre recombinase technology, NIR-light stimulation successfully performs genome engineering resulting in the formation of dendritic-spine-like structures of hippocampal neurons.


Assuntos
Complexos de Coordenação/química , Corantes Fluorescentes/química , Hidrogéis/química , Osmio/química , Perileno/química , Genoma , Raios Infravermelhos , Cinética , Micelas , Estrutura Molecular , Optogenética/métodos , Oxigênio/química , Fótons , Poloxâmero/química , Relação Estrutura-Atividade , Termodinâmica
6.
Chemistry ; 25(59): 13523-13530, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31283853

RESUMO

Self-assembling peptides that are capable of adopting ß-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a ß-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted ß-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.


Assuntos
Adesivos/química , Glicinérgicos/metabolismo , Glicina/química , Peptídeos/química , Glicinérgicos/química , Hidrogéis/química , Simulação de Dinâmica Molecular , Nanofibras/química , Reologia
7.
Development ; 144(18): 3303-3314, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28928282

RESUMO

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance.


Assuntos
Divisão Celular , Quinase 1 do Ponto de Checagem/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Animais , Morte Celular , Hipóxia Celular , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1 , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Glucose/deficiência , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese , Oxigênio , Proteína do Retinoblastoma/metabolismo , Fase S , Transdução de Sinais , Acidente Vascular Cerebral/patologia
8.
Adv Healthc Mater ; 6(11)2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28488337

RESUMO

Ischemic brain stroke is caused by blood flow interruption, leading to focal ischemia, neuron death, and motor, sensory, and/or cognitive dysfunctions. Angiogenesis, neovascularization from existing blood vessel, is essential for tissue growth and repair. Proangiogenic therapy for stroke is promising for preventing excess neuron death and improving functional recovery. Vascular endothelial growth factor (VEGF) is a critical factor for angiogenesis by promoting the proliferation, the survival, and the migration of endothelial cells. Here, angiogenic biomaterials to support injured brain regeneration are developed. Porous laminin (LN)-rich sponge (LN-sponge), on which histidine-tagged VEGF (VEGF-Histag) is immobilized via affinity interaction is developed. In an in vivo mouse stroke model, transplanting VEGF-Histag-LN-sponge produces remarkably stronger angiogenic activity than transplanting LN-sponge with soluble VEGF. The findings indicate that using affinity interactions to immobilize VEGF is a practical approach for developing angiogenic biomaterials for regenerating the injured brain.


Assuntos
Isquemia Encefálica , Proteínas Imobilizadas , Laminina , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Modelos Animais de Doenças , Implantes de Medicamento/química , Implantes de Medicamento/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Laminina/química , Laminina/farmacologia , Camundongos , Porosidade , Fator A de Crescimento do Endotélio Vascular/farmacologia
9.
Development ; 140(11): 2310-20, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23615279

RESUMO

Cell cycle dysregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via the Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107 and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found in mouse that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and proliferated when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb(-/-); p107(-/-); p130(-/-) (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated the DNA double-strand break (DSB) repair pathway without increasing trimethylation at lysine 20 of histone H4 (H4K20), which has a role in protection against DNA damage. The activation of the DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly born cortical neurons from progenitors become epigenetically protected from DNA damage and cell division in an Rb family-dependent manner.


Assuntos
Córtex Cerebral/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Córtex Cerebral/embriologia , Dano ao DNA , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Camundongos , Camundongos Knockout , Reparo de DNA por Recombinação , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/metabolismo , Proteína p130 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/metabolismo
10.
Invest Ophthalmol Vis Sci ; 53(2): 1033-40, 2012 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-22302105

RESUMO

PURPOSE: To screen for disease-causing mutations in the Eyes shut homolog (EYS) gene in Japanese patients with retinitis pigmentosa (RP). Methods. Blood samples were obtained from 68 RP patients and 68 controls. Genomic DNA was extracted from the blood samples and used for screening of mutations in the coding exons by direct sequencing. Each patient underwent a detailed clinical examination. RESULTS: Nine nucleotide sequence variations causing amino acid changes were observed in homozygous or heterozygous alleles in 26 patients but not in 68 controls. Seven truncating mutations were found in 21 (32.8%) of 64 patients with nonsyndromic RP composed of 23 autosomal recessive RP (arRP) and 41 sporadic cases. The most abundant mutation was p.S1653Kfs*2, which was generated by a single adenine insertion into exon 26 (c.4957dupA) and was carried by 15 patients. The mutation p.Y2935*, produced by a single nucleotide substitution (c.8805C>A) in the last exon, was carried by five patients. These two truncating mutations were probably founder mutations because each was carried by the particular haplotype. The patients with homozygous or compound heterozygous truncating mutations showed a severe decline in visual acuity, whereas those with a single truncating mutation showed a mild decline. CONCLUSIONS: One-third of Japanese patients with nonsyndromic arRP carried probable pathogenic mutations in the EYS gene, including two founder mutations. Because the genotype was correlated with the phenotype, genotyping in the EYS gene could be a valuable tool for predicting long-term prognoses of Japanese patients with arRP and thus could be useful for genetic counseling and future gene therapy.


Assuntos
Éxons/genética , Proteínas do Olho/genética , Mutação/genética , Retinose Pigmentar/genética , Adulto , Idoso , Povo Asiático/genética , Estudos de Casos e Controles , Feminino , Genes Recessivos/genética , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade
11.
Invest Ophthalmol Vis Sci ; 52(9): 6662-70, 2011 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-21697133

RESUMO

PURPOSE. To collect an entire set of full-length cDNA clones derived from human retina-derived cell lines and to identify full-length transcripts for retinal preferentially expressed genes. METHODS. The full-length cDNA libraries were constructed from a retinoblastoma cell line, Y79, and a retinal pigment epithelium cell line, ARPE-19, using the vector-capping method, which generates a genuine full-length cDNA. By single-pass sequencing of the 5'-end of cDNA clones and subsequent mapping to the human genome, the authors determined their transcriptional start sites and annotated the cDNA clones. RESULTS. Of the 23,616 clones isolated from Y79-derived cDNA libraries, 19,229 full-length cDNA clones were identified and classified into 4808 genes, including genes of >10 kbp. Of the 7067 genes obtained from the Y79 and ARPE-19 libraries, the authors selected 72 genes that were preferentially expressed in the eye, of which 131 clones corresponding to 57 genes were fully sequenced. As a result, we discovered many variants that were produced by different transcriptional start sites, alternative splicing, and alternative polyadenylation. CONCLUSIONS. The bias-free, full-length cDNA libraries constructed using the vector-capping method were shown to be useful for collecting an entire set of full-length cDNA clones for these retinal cell lines. Full-length transcriptome analysis of these cDNA libraries revealed that there were, unexpectedly, many transcript variants for each gene, indicating that obtaining the full-length cDNA for each variant is indispensable for analyzing its function. The full-length cDNA clones (approximately 80,000 clones each for ARPE-19 and Y79) will be useful as a resource for investigating the human retina.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/fisiologia , Expressão Gênica/fisiologia , Capuzes de RNA/genética , Neoplasias da Retina/genética , Epitélio Pigmentado da Retina/metabolismo , Retinoblastoma/genética , Linhagem Celular Tumoral , Clonagem Molecular , Biblioteca Gênica , Genes Neoplásicos , Vetores Genéticos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plasmídeos , RNA Mensageiro/análise , Transcrição Gênica
12.
Methods Mol Biol ; 729: 53-70, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21365483

RESUMO

Full-length complementary DNAs (cDNAs) are an essential resource for functional genomics. Recently, we have developed a simple and efficient method for preparing a full-length cDNA library from a small amount of total RNA, named the "vector-capping" method. The biggest advantage of this method is that the intactness of the cDNA can be assured by the presence of dG at the 5' end of the full-length cDNA. Furthermore, the cDNA library represents the mRNA population in the cell owing to a bias-free procedure. In this chapter, we describe not only the protocol for preparing the library but also the points for analyzing the 5'-end sequence of the obtained cDNA.


Assuntos
DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Análise de Sequência de DNA/métodos , Sequência de Bases , DNA Complementar/metabolismo , Vetores Genéticos/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/análise
13.
Mol Vis ; 15: 482-94, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19262745

RESUMO

PURPOSE: The aim of this study was to characterize the arylsulfatase I (ARSI) gene that has been shown to be preferentially expressed in the human retinal pigment epithelium cell line ARPE-19 and to propose it as a candidate gene responsible for inherited eye diseases such as retinitis pigmentosa (RP). METHODS: Full-length cDNA clones encoding ARSI, arylsulfatase A (ARSA), and sulfatase modifying factor 1 (SUMF1) were isolated from ARPE-19 cDNA libraries constructed using the vector-capping method. The expression vectors for their FLAG-tagged proteins were transfected into ARPE-19 cells, and the expression products were characterized by western blot analysis and arylsulfatase assay. The entire region of the ARSI gene locus was sequenced using the genomic DNA samples of 68 RP patients. RESULTS: Transiently produced ARSI-FLAG was localized to the endoplasmic reticulum and was detected in the cellular fraction and the medium. When ARSI-FLAG and SUMF1-FLAG were coexpressed, the conditioned medium of the transfected cells showed arylsulfatase activity at a range of neutral pH. No mutation was found in the ARSI gene locus of the RP patients examined. CONCLUSIONS: ARSI may be a secreted sulfatase and may function in the extracellular space. Although ARSI may not be a causative gene for lysosomal storage diseases, preferentially expressed in the eye, ARSI would be a candidate gene causing inherited eye diseases for future mutation screening.


Assuntos
Arilsulfatases/genética , Epitélio Pigmentado da Retina/enzimologia , Substituição de Aminoácidos , Arilsulfatases/metabolismo , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Análise Mutacional de DNA , Primers do DNA/genética , DNA Complementar/genética , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Transfecção
14.
DNA Res ; 15(3): 123-36, 2008 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-18487259

RESUMO

Recently, we have developed a vector-capping method for constructing a full-length cDNA library. In the present study, we performed in-depth analysis of the vector-capped cDNA library prepared from a single type of cell. As a result of single-pass sequencing analysis of 24,000 clones randomly isolated from the unamplified library, we identified 19,951 full-length cDNA clones whose intactness was confirmed by the presence of an additional G at their 5' end. The full-length cDNA content was >95%. Mapping these sequences to the human genome, we identified 4,513 transcriptional units that include 36 antisense transcripts against known genes. Comparison of the frequencies of abundant clones showed that the expression profiles of different libraries, including the distribution of transcriptional start sites (TSSs), were reproducible. The analysis of long-sized cDNAs showed that this library contained many cDNAs with a long-sized insert up to 11,199 bp of golgin B, including multiple slicing variants for filamin A and filamin B. These results suggest that the size-unbiased full-length cDNA library constructed using the vector-capping method will be an ideal resource for fine expression profiling of transcriptional variants with alternative TSSs and alternative splicing.


Assuntos
Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Vetores Genéticos , Capuzes de RNA/genética , RNA Mensageiro/análise , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , Bases de Dados de Ácidos Nucleicos , Dosagem de Genes , Vetores Genéticos/fisiologia , Humanos , Capuzes de RNA/química , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , Reprodutibilidade dos Testes , Sítio de Iniciação de Transcrição/fisiologia
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