RESUMO
Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Jatropha/enzimologia , Jatropha/química , Hemiterpenos/genética , Hemiterpenos/metabolismo , Filogenia , RNA/isolamento & purificação , Expressão Gênica , Cloroplastos , Southern Blotting , Clonagem Molecular , DNA Complementar/síntese química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This in vivo study investigated the time of degradation of root filling material (trans 1,4-polyisoprene) retrieved from endodontically treated teeth and correlated the occurrence of degradation with the longevity of endodontics. Thirty-six root-filled teeth with different filling times (2 to 30 years) and with and without periapical lesions were selected. All teeth presented clinical indication for root canal retreatment. The association among filling time, presence of periapical lesion and root filling material degradation was investigated. Root filling samples were retrieved from the root canals using a Hedströ m file without solvent. The trans 1,4-polyisoprene was isolated by root filling solubilization in chloroform followed by filtration and centrifugation. GPC and FT-IR were the analytical techniques utilized. Degradation of trans 1,4-polyisoprene occurred with time, as a slow process. It is an oxidative process, and production of carboxyl and hydroxyl groups in the residual polymer were observed. Statistically significant decrease of molar mass was observed after 5 (p=0.0001) and 15 (p=0.01) years in teeth with and without periapical lesion, respectively. Bacteria participated in polymer degradation. Gutta-percha aging was proven an important factor for the long-term success of endodontic treatment. The findings of the present study showed that, after 15 years, polymer weight loss may decrease the capacity of the filling mass to seal the root canal space and prevent re-infection, thus compromising significantly the longevity of root canal therapy.
Este estudo in vivo avaliou a degradação do material obturador e a influência deste fator na longevidade do tratamento endodôntico. Foram selecionados 36 pacientes (3-30 anos) com canais tratados endodonticamente, com e sem lesões periapicais, e indicação de retratamento endodôntico. Foi investigada a associação entre o tempo de tratamento, presença de lesão periapical e a degradação do material obturador. O material obturador foi removido com uma lima Hedströ em sem uso de solvente. O polímero trans 1,4- poliisopreno foi isolado do material obturador através de solubilização em clorofórmio, seguido de filtragem e centrifugação. GPC e FT-IR foram os métodos analíticos utilizados. A degradação do trans 1,4- poliisopreno foi observada com o tempo, sendo um processo lento e oxidativo, com formação de grupos carboxílicos e hidroxilas no polímero residual. Após 5 (p=0,0001) e 15 (p=0,01) anos, em dentes com e sem lesões periapicais, respectivamente, houve decréscimos significantes na massa molar do material obturador. A infecção bacteriana participa no processo de degradação do polímero. O envelhecimento da guta-percha é um fator que influencia o sucesso a longo prazo do tratamento endodôntico. Após 15 anos, a longevidade do tratamento pode ser significantemente afetada pela redução da capacidade de selamento causada pela perda de massa molar do polímero, permitindo a reinfecção do sistema de canais radiculares.
