RESUMEN
The restoration of body contours as shaped by adipose tissue remains a clinical challenge specifically in patients who have experienced loss of contour due to trauma, surgical removal of tumours or congenital abnormalities. We have developed a novel macro-microporous biomaterial for use in soft tissue re-bulking and augmentation. Alginate beads provided the pore template for the construct. Incorporation, and subsequent dissolution, of the beads within a 7 % (w/v) gelatin matrix, produced a highly porous scaffold with an average pore size of 2.01 ± 0.08 mm. The ability of this scaffold to support the in vitro growth and differentiation of human adipose-derived stem cells (ADSCs) was then investigated. Histological analysis confirmed that the scaffold itself provided a suitable environment to support the growth of ADSCs on the scaffold walls. When delivered into the macropores in a fibrin hydrogel, ADSCs proliferated and filled the pores. In addition, ADSCs could readily be differentiated along the adipogenic lineage. These results therefore describe a novel scaffold that can support the proliferation and delivery of ADSCs. The scaffold is the first stage in developing a clinical alternative to current treatment methods for soft tissue reconstruction.
Asunto(s)
Tejido Adiposo/citología , Células Madre Adultas/trasplante , Gelatina/química , Gelatina/síntesis química , Regeneración Tisular Dirigida , Andamios del Tejido/química , Células Madre Adultas/citología , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/fisiología , Alginatos/química , Alginatos/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Gelatina/farmacología , Ácido Glucurónico/química , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/química , Ácidos Hexurónicos/farmacología , Humanos , Ensayo de Materiales , Microtecnología/métodos , PorosidadRESUMEN
Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.
Asunto(s)
Daño del ADN/efectos de los fármacos , ADN Polimerasa I/metabolismo , Reparación del ADN/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Alquilantes/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/efectos de la radiación , ADN Polimerasa I/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Inestabilidad Genómica , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fase S/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Rayos UltravioletaRESUMEN
Bi-directionality is a common feature observed for genomic replication for all three phylogenetic kingdoms: Eubacteria, Archaea, and Eukaryotes. A consequence of bi-directional replication, where the two replication forks initiated at an origin move away from each other, is that the replication termination will occur at positions away from the origin sequence(s). The replication termination processes are therefore physically and mechanistically dissociated from the replication initiation. The replication machinery is a highly processive complex that in short time copies huge numbers of bases while competing for the DNA substrate with histones, transcription factors, and other DNA-binding proteins. Importantly, the replication machinery generally wins out; meanwhile, when converging forks meet termination occurs, thus preventing over-replication and genetic instability. Very different scenarios for the replication termination processes have been described for the three phylogenetic kingdoms. In eubacterial genomes replication termination is site specific, while in archaea and eukaryotes termination is thought to occur randomly within zones where converging replication forks meet. However, a few site-specific replication barrier elements that mediate replication termination have been described in eukaryotes. This review gives an overview about what is known about replication termination, with a focus on these natural site-specific replication termination sites.
Asunto(s)
Replicación del ADN/fisiología , Archaea/genética , Archaea/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión , Replicación del ADN/genética , ADN de Archaea/biosíntesis , ADN de Archaea/genética , ADN Bacteriano/biosíntesis , ADN Bacteriano/genética , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Ribosómico/biosíntesis , ADN Ribosómico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of "slow-moving" Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Replicación del ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genoma Fúngico , Datos de Secuencia Molecular , Mutación , Fenotipo , Plásmidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Structure chromosome (SMC) proteins organize the core of cohesin, condensin and Smc5-Smc6 complexes. The Smc5-Smc6 complex is required for DNA repair, as well as having another essential but enigmatic function. Here, we generated conditional mutants of SMC5 and SMC6 in budding yeast, in which the essential function was affected. We show that mutant smc5-6 and smc6-9 cells undergo an aberrant mitosis in which chromosome segregation of repetitive regions is impaired; this leads to DNA damage and RAD9-dependent activation of the Rad53 protein kinase. Consistent with a requirement for the segregation of repetitive regions, Smc5 and Smc6 proteins are enriched at ribosomal DNA (rDNA) and at some telomeres. We show that, following Smc5-Smc6 inactivation, metaphase-arrested cells show increased levels of X-shaped DNA (Holliday junctions) at the rDNA locus. Furthermore, deletion of RAD52 partially suppresses the temperature sensitivity of smc5-6 and smc6-9 mutants. We also present evidence showing that the rDNA segregation defects of smc5/smc6 mutants are mechanistically different from those previously observed for condensin mutants. These results point towards a role for the Smc5-Smc6 complex in preventing the formation of sister chromatid junctions, thereby ensuring the correct partitioning of chromosomes during anaphase.
