RESUMEN
Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.
Asunto(s)
Biopolímeros/química , Colágeno/química , Elastina/química , Andamios del Tejido/química , Animales , Biopolímeros/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno/farmacología , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Ingeniería de TejidosRESUMEN
CYP3A4, a cytochrome P450 enzyme regulated by the nuclear receptor PXR, is involved in most of the drug metabolizing pathways. Studying the regulation/induction of CYP3A4 and PXR is critical in toxicology and drug-drug interaction (DDI) studies. Primary human hepatocytes constitute the preferred in vitro platform for drug development efforts. However, they are expensive, scarce and heterogeneous. Hepatic cell lines, such as Huh7, could provide a cost-effective alternative, however, they express negligible amounts of CYP450s and PXR. In this study, we show that dinaciclib, a potent cyclin dependent kinase inhibitor, significantly increases the basal CYP3A4 and PXR levels in 24 hours. We also demonstrated that matured Huh7s can be used for drug induction studies, where CYP3A4, CYP1A2, CYP2C9, and CYP2C19 inductions were achieved following rifampicin treatment. More importantly, through a direct demonstration using amiodarone and rifampicin as model drugs, we showed that matured Huh7s present a suitable platform for DDI studies.
Asunto(s)
Amiodarona/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Hígado/metabolismo , Receptor X de Pregnano/metabolismo , Rifampin/farmacología , Línea Celular Tumoral , Inducción Enzimática/efectos de los fármacos , Humanos , Hígado/citologíaRESUMEN
Foldback priming at DNA double-stranded breaks is one mechanism proposed to initiate palindromic gene amplification, a common feature of cancer cells. Here, we show that small (5-9 bp) inverted repeats drive the formation of large palindromic duplications, the major class of chromosomal rearrangements recovered from yeast cells lacking Sae2 or the Mre11 nuclease. RPA dysfunction increased the frequency of palindromic duplications in Sae2 or Mre11 nuclease-deficient cells by â¼ 1,000-fold, consistent with intra-strand annealing to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. The palindromic duplications were frequently associated with duplication of a second chromosome region bounded by a repeated sequence and a telomere, suggesting the dicentric chromosome breaks and repairs by recombination between dispersed repeats to acquire a telomere. We propose secondary structures within single-stranded DNA are potent instigators of genome instability, and RPA and Mre11-Sae2 play important roles in preventing their formation and propagation, respectively.
Asunto(s)
Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Amplificación de Genes , Secuencias Invertidas Repetidas , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Roturas del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Exodesoxirribonucleasas/genética , Inestabilidad Genómica/genética , Isocromosomas/genética , Proteína de Replicación A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Soluble guanylate cyclase (sGC) serves as a receptor for the signaling agent nitric oxide (NO). sGC synthesis of cGMP is regulated by NO, GTP, ATP, and allosteric activators such as YC-1. The guanylate cyclase activity and adenylate cyclase activity of full-length sGC and the sGC catalytic domain constructs (alpha1(cat)beta1(cat)) are reported here. ATP is a mixed-type inhibitor of cGMP production for both sGC and alpha1(cat)beta1(cat), indicating that the C-terminus of sGC contains an allosteric nucleotide binding site. YC-1 did not activate alpha1(cat)beta1(cat) or compete with ATP inhibition of cGMP synthesis, which suggests that YC-1 and ATP bind to distinct sites. alpha1(cat)beta1(cat) and NO-stimulated sGC also synthesize cAMP, but this activity is inhibited by ATP via noncompetitive substrate inhibition and by GTP via mixed-type inhibition. Additionally, the adenylate cyclase activity of purified sGC was inhibited by PC12 lysate, suggesting that an intracellular small molecule or protein regulates this activity in vivo.