RESUMO
Cathepsin A (CTSA) is a multifunctional lysosomal enzyme, and its hereditary defect causes an autosomal recessive disorder called galactosialidosis. In a certain number of galactosialidosis patients, a base substitution from adenine to guanine is observed at the +3 position of the 7th intron (IVS7 +3a>g) of the CTSA gene. With this mutation, a splicing error occurs; and consequently mRNA lacking the 7th exon is produced. This skipping of exon 7 causes a frame shift of the transcripts, resulting in a non-functional CTSA protein and hence galactosialidosis. This mutation seems to make the interaction between the 5'-splice site of intron 7 of pre-mRNA and U1 small nuclear RNA (U1 snRNA) much weaker. In the present study, to produce properly spliced mRNA from the CTSA gene harboring this IVS7 +3a>g mutation, we examined the possible usefulness of modified U1 snRNA that could interact with the mutated 5'-splice site. Toward this goal, we first prepared a model system using a mutant CTSA mini gene plasmid for delivery into HeLa cells. Then, we examined the effectiveness of modified U1 snRNA on the formation of properly spliced mRNA from this mutant CTSA mini gene. As a result, we succeeded in obtaining improved formation of properly spliced CTSA mRNA. Our results suggest the usefulness of modified U1 snRNA for rescue from exon 7 skipping caused by the IVS7 +3a>g mutation of the CTSA gene.
Assuntos
Catepsina A/genética , Éxons/genética , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/genética , Linhagem Celular Tumoral , Células HeLa , Humanos , Íntrons/genética , Mutação/genética , Precursores de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genéticaRESUMO
A sugarless layer that provides protection against moisture is formed on tablets when a coating solution comprising mannitol and poly(vinyl alcohol) (PVA) is applied. The objective of this study is to investigate the relationship between the formation of such a sugarless layer and the resulting hygroscopic properties in order to derive an appropriate sugarless coating. The hygroscopicity of the sugarless layer is shown to be strongly affected by the addition of PVA, and has the lowest at concentration ratios between 15:2.5 and 15:4 (w/w) of mannitol and PVA. The polymorphic form of mannitol is different in formulations with different mannitol:PVA concentration ratios. Mannitol occurs in the α-form at mannitol:PVA concentration ratios between 15:1 and 15:4 (w/w). Moreover, PVA affects the molecular motions in the region associated with the OH stretch, OH deformation, and CH2 wag of mannitol. In particular, the molecular motions change considerably at mannitol:PVA concentration ratio of 15:2.5 and 15:4 (w/w). In addition, the surface state of the sugarless layer depends on the amount of PVA added, and exhibits the smoothest surface at a mannitol:PVA concentration ratio between 15:2.5 and 15:4 (w/w). Thus, the hygroscopicity is related to the surface states of the sugarless layer, which, in turn, is affected by the change in the molecular motions of mannitol due to the interactions between mannitol and PVA.