RESUMO
Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Young's modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.
Assuntos
Materiais Biocompatíveis , Colágeno/química , Elastina/química , Proteínas de Insetos/química , Engenharia de Proteínas , Sequência de Bases , Western Blotting , Dicroísmo Circular , Colágeno/síntese química , Colágeno/genética , Primers do DNA , Elastina/síntese química , Elastina/genética , Proteínas de Insetos/síntese química , Proteínas de Insetos/genética , Microscopia de Força Atômica , Reação em Cadeia da Polimerase , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
BACKGROUND: Comprehensive analyses have recently been performed on many human cancer tissues, leading to the identification of a number of mutated genes but providing no information on the variety of mutations present in each of them. This information is of interest to understand the possible origin of gene mutations that cause tumors. METHODOLOGY/PRINCIPAL FINDINGS: We have analyzed the sequence heterogeneity of the transcripts of the human HPRT and G6PD single copy genes that are not considered tumor markers. Analyses have been performed on different colon cancers and on the nearby histologically normal tissues of two male patients. Several copies of each cDNA, which were produced by cloning the RT-PCR-amplified fragments of the specific mRNA, have been sequenced. Similar analyses have been performed on blood samples of two ostensibly healthy males as reference controls. The sequence heterogeneity of the HPRT and G6PD genes was also determined on DNA from tumor tissues. The employed analytical approach revealed the presence of low-frequency mutations not detectable by other procedures. The results show that genetic heterogeneity is detectable in HPRT and G6PD transcripts in both tumors and nearby healthy tissues of the two studied colon tumors. Similar frequencies of mutations are observed in patient genomic DNA, indicating that mutations have a somatic origin. HPRT transcripts show genetic heterogeneity also in healthy individuals, in agreement with previous results on human T-cells, while G6PD transcript heterogeneity is a characteristic of the patient tissues. Interestingly, data on TP53 show little, if any, heterogeneity in the same tissues. CONCLUSIONS/SIGNIFICANCE: These findings show that genetic heterogeneity is a peculiarity not only of cancer cells but also of the normal tissue where a tumor arises.
Assuntos
Colo/metabolismo , Neoplasias Colorretais/genética , Instabilidade Genômica , Mutação , Idoso , Neoplasias Colorretais/patologia , DNA Complementar/genética , DNA de Neoplasias/genética , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Glucosefosfato Desidrogenase/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Íntrons/genética , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/genética , Adulto JovemRESUMO
DNA methylation plays a central role in the control of gene expression during development and cell differentiation, thus DNA methylation and demethylation processes are expected to be strictly regulated during these events. We have explored the expression levels of the genes encoding DNA methylases, methyl-CpG binding proteins and demethylases during in vitro differentiation of human carcinoma colon cells (CaCO-2) used as a model system. The results show that the global DNA methylation pattern remains constant during CaCO-2 cells differentiation indicating that required genome methylation pattern in cell differentiation was already established in the seeded cells. On the contrary, the timing of expression of several of the explored genes is tightly regulated, suggesting they are involved in the regulation of the differentiation program. In particular, the timing of expression of DNMT3b and of MBD2b and 5-MCDG shows two peaks not observed in the time courses of the expression of other genes belonging to the same families. These events, not dependent on the cell cycle synchronization, have apparently no significant impact on the overall methylation status of the genome.