RESUMO
To modify the flavor properties of tomato fruits, cucumber fatty acid hydroperoxide lyase (HPL), which can act on 9-hydroperoxides of fatty acids to form volatile C9-aldehydes, was introduced to tomato plants. Through enzyme assay, high activity of the introduced HPL could be found in either the leaves or fruits of transgenic tomatoes; however, the composition of volatile short-chain aldehydes and alcohols in the transgenic tomato fruits was little modified. This was unexpected because tomato fruits have high lipoxygenase activity to form 9-hydroperoxides. When linoleic acid was added to a crude homogenate prepared from the transgenic tomato fruits, a high amount of C9-aldehyde was formed, but the amount of C6-aldehyde was almost equivalent to that in nontransgenic tomatoes. Through quantification of fatty acid hydroperoxides, it has been revealed that 13-hydroperoxides of fatty acids are preferably formed from endogenous substrate, whereas 9-hydroperoxides are formed from fatty acids added exogenously. From these observations, possible mechanisms to regulate metabolic flow of the lyase pathway are discussed.
Assuntos
Hidroliases/genética , Solanum lycopersicum/genética , Aldeídos/metabolismo , Hidroliases/metabolismo , Solanum lycopersicum/enzimologia , TransgenesRESUMO
Although the restenosis rate of coronary stenting is generally 10% to 20%, it can go as high as 60% in patients with diabetes or complex lesions. Currently, the only effective treatment for restenosis is brachytherapy. Drug-eluting stents may be the way to prevent restenosis that cardiologists have been seeking: the drug-coated stents are simple to use and help prevent negative remodeling and the intimal hyperplasia caused by stenting. In studies comparing sirolimus-coated and bare-metal stents, the sirolimus-coated stents resulted in less smooth muscle cell colonization, minimal intimal hyperplasia, and no edge effect; moreover, no adverse clinical events were reported. Currently ongoing, multicenter clinical trials of drug-eluting stents may soon come up with the answers that cardiologists have been hoping for.
Assuntos
Angioplastia Coronária com Balão/instrumentação , Estenose Coronária/terapia , Sirolimo/farmacologia , Stents , Angioplastia Coronária com Balão/métodos , Materiais Revestidos Biocompatíveis , Angiografia Coronária , Estenose Coronária/diagnóstico por imagem , Desenho de Equipamento , Segurança de Equipamentos , Feminino , Humanos , Masculino , Prevenção Secundária , Sensibilidade e Especificidade , Grau de Desobstrução VascularRESUMO
Fatty acid hydroperoxide lyase (HPL) is a novel P-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction-based cloning strategy, we isolated two HPL-related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13-hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9- and 13-hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.
Assuntos
Aldeído Liases/metabolismo , Cucumis sativus/enzimologia , Sistema Enzimático do Citocromo P-450 , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/isolamento & purificação , Aldeídos/metabolismo , Cromatografia Líquida de Alta Pressão , Mutação da Fase de Leitura/genética , Hidroliases , Peróxido de Hidrogênio/metabolismo , Hipocótilo/enzimologia , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Filogenia , Pseudogenes/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Fatty acid hydroperoxide lyase (HPL) is a member of a novel subfamily of cytochrome P450 and catalyzes a cleavage reaction of fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. A cDNA encoding tomato fruit HPL (LeHPL) was obtained. An active LeHPL was expressed in E. coli and purified. It showed highest activity against the 13-hydroperoxide of linolenic acid, followed by that of linoleic acid. 9-Hydroperoxides were poor substrates. The absorption spectrum of the purified LeHPL in the native form was similar to that of most P450s although a CO-adduct having a lambda max at 450 nm could not be obtained. LeHPL activity is reversibly inhibited by nordihydroguaiaretic acid, while salicylic acid irreversibly inhibited it. LeHPL is kinetically inactivated by fatty acid hydroperoxides, especially 9-hydroperoxides. The inactivation is prevented by inhibitors of LeHPL. Thus, HPL catalytic activity is thought to be essential to its inactivation. During the inactivation, an abolition of the Soret band was evident, indicating that inactivation is caused mainly by degradation of the prosthetic heme in LeHPL.
Assuntos
Aldeído Liases/genética , Sistema Enzimático do Citocromo P-450 , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Escherichia coli/genética , Heme/química , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Fatty acid hydroperoxide lyase (HPOL), an enzyme of the octadecanoid pathway that forms carbon-6 aldehydes such as n-hexanal or (Z)-3-hexenal, was cloned from Arabidopsis thaliana as a full-length cDNA. The HPOL activity obtained by expressing the cDNA in Escherichia coli formed n-hexanal from linoleic acid 13-hydroperoxide, whereas linoleic acid 9-hydroperoxide was not a substrate for the enzyme. The HPOL mRNA is expressed at low level in leaves; however, its accumulation can be found in the inflorescence. Wounding or methyl jasmonate treatments increase the mRNA level in leaves. These results indicate that the HPOL gene is up-regulated in leaves in response to wounding and that the enzyme may be an active component of the octadecanoid defense response.