RESUMO
We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.
Assuntos
Nucleosídeo NM23 Difosfato Quinases/farmacologia , Células-Tronco/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ligantes , MicroRNAs/genética , MicroRNAs/metabolismo , Mucina-1/imunologia , Mucina-1/metabolismo , Nucleosídeo NM23 Difosfato Quinases/química , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Gold nanoparticles hold great promise for studying protein-protein interactions because of their intrinsic optical properties. Pink when in a homogeneous suspension, the solution turns blue-gray when particles are drawn close together, for example, when immobilized proteins specifically interact with each other. However, the nanoparticle stability, size, and method of protein attachment contribute to the unreliable outcome of current assays. To overcome these hurdles, we developed novel and reliable methods first to synthesize homogenous particles of optimal diameter and second to apply a heterologous NTA-Ni-SAM coating for controlled orientation and optimal presentation of histidine-tagged proteins. Both methods were proven to greatly enhance assay sensitivity and specificity by increasing the signal and minimizing the nonspecific binding. Our assay reproducibly detected known protein-protein interactions and unambiguously identified small molecules that inhibited them. We believe our gold nanoparticle bioassay is a versatile and trustworthy new platform for analyzing protein-protein interactions and high-throughput screening of small-molecule inhibitors.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Proteínas/química , Avaliação de Medicamentos , Ensaios de Triagem em Larga Escala , Histidina/genética , Histidina/metabolismo , Mucina-1/química , Mucina-1/genética , Mucina-1/metabolismo , Ácido Nitrilotriacético/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismoRESUMO
We describe here the synthesis and properties of A-T rich DNA containing covalently bound water mimics located in the DNA minor groove.
Assuntos
Sequência Rica em At , Materiais Biomiméticos/química , DNA/química , DNA/genética , Água/química , Adenina/química , Sequência de Bases , Conformação de Ácido NucleicoRESUMO
The purine nucleoside 2,6-diaminopurine-2'-deoxyriboside is prepared by the direct glycosylation of the 2,6-bis(tetramethylsuccinimide) derivative of the parent purine heterocycle 4 with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride 5 using the sodium salt method. 2'-Deoxyisoguanosine is prepared from 2,6-diaminopurine by a five-step procedure. The purine heterocycle isoguanine is prepared by selective diazotization of 2,6-diaminopurine and then converted to the N9-trityl derivative to increase solubility. After silylation of the O(2)-carbonyl with TMSCl, the N(6)-amino group is protected as the tetramethylsuccinimide (M(4)SI). The O(2)-carbonyl is protected as the DPC derivative, and the trityl group is removed. The resulting product is glycosylated in good yield to generate fully protected 2'-deoxyisoguanosine.
Assuntos
2-Aminopurina/análogos & derivados , Guanina/síntese química , Guanosina/síntese química , Nucleosídeos/síntese química , Nucleosídeos de Purina/síntese química , 2-Aminopurina/síntese química , 2-Aminopurina/química , Adenosina , Glicosilação , Guanina/química , Guanosina/química , Estrutura Molecular , Nucleosídeos/química , Nucleosídeos de Purina/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Tetramethylsuccinic anhydride can be used to protect the exocyclic amine of 6-aminopurine derivatives by forming the corresponding tetramethysuccinimide. X-ray crystallography confirms that the imide carbonyl and the methyl groups are positioned to sterically block the N7 nitrogen so that glycosylations occur with very high regiochemical control at N9. This approach is particularly effective for 3-substituted purines where the substituent tends to block access to N9 and inhibit glycosylation at that site.
