RESUMO
Clarifying the principles of proportioning optimization for brittle transparent rock-like specimens with differential fracture structures is crucial for the visualization study of the internal fracture and seepage evolution mechanisms in rock masses. This study, utilizing orthogonal experimental methods, uncovers the influence mechanisms, extents, and patterns by which the ratios of resin, hardener, and accelerator, along with the freezing duration, impact the mechanical characteristics of transparent rock-like specimens. Notably, it was observed that as the accelerator ratio and freezing time are increased, there's a general decline in the uniaxial compressive strength, tensile strength, and elastic modulus of the specimens. In contrast, an increase in the hardener ratio initially leads to an enhancement in these mechanical properties, followed by a subsequent decrease. Under uniaxial compressive loading, the specimens exhibit four typical modes of failure: bursting failure, splitting failure, single inclined plane failure, and bulging failure. As the hardener and accelerator ratios increase, the mode of failure gradually shifts from bulging to bursting, with freezing time having a minor overall impact on the evolution of failure modes. The study proposes a method for inducing random three-dimensional closed fractures within the specimens and further clarifies the principles for optimizing the proportions of specimens with different fracture structures, such as intact, embedded regular, and random three-dimensional fractures. This research facilitates the in-depth application of transparent rock-like materials in various scenarios and provides theoretical guidance and technical support for visualizing the evolution of fracture and seepage characteristics within the fractured rock mass.
RESUMO
The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.
Assuntos
Apoptose , Caspases/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspase 3 , Caspase 9 , Inibidores de Caspase , Caspases/química , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
In sequencing the beta3 chain of laminin 5 mRNA from LNCaP cells, we observed three different human cDNA clones (XM_001716, NM_000228 and L25541) in the GenBank that identified different sequences in the untranslated regions (UTR). XM_001716 and NM_000228 are almost identical cDNA clones with approximately 99% homology. However, they are quite different from L25541 in both the 5' UTR and the 3' UTR. Development of a PCR assay to specifically detect two of these different forms of the message led to the observation that they were differentially expressed in various cell lines. The message designated B3A (NM_000228, and XM_001716) was absent in LNCaP and MCF7 and greatly reduced in PC3-N, but was present in eight other epithelial cell lines. B3B (L25541) was present in all cell lines studied. The cell lines that failed to express the B3A form also failed to express the protein based on both immunoblotting and immunohistochemical analysis. It appears from this data that there are two isoforms of the beta3 mRNA, and that the 5' UTRs of the mRNAs play an important role in regulating translation of the beta3 protein. Since laminin 5 is lost in prostate carcinoma, the mechanism of control that results in the translation of the two forms of message may be important in tumorigenesis.