RESUMO
Xanthohumol, isoxanthohumol, and 8-prenylnaringenin in beer, hop and hop pellet samples were analyzed by HPLC using an InertSustain phenyl column and the mobile phase containing 40% methanol and 12% 2-propanol. Fractions of isoxanthohumol and 8-prenylnaringenin obtained by the above HPLC were separately collected. Isoxanthohumol and 8-prenylnaringenin were enantioseparated by HPLC using a Chiralcel OD-H column with a mobile phase composed of hexane-ethanol (90:10, v/v) and a Chiralpak AD-RH column with a mobile phase composed of methanol-2-propanol-water (40:20:40, v/v/v), respectively. Isoxanthohumol and 8-prenylnaringenin from beer, hop and hop pellet samples were found to be present in a racemic mixture. This can be explained by the fact that the two analytes were produced by a nonenzymatic process. The effects of boiling conditions on the conversion of xanthohumol into isoxanthohumol were also studied. A higher concentration of ethanol in heating solvent resulted in a decrease in the conversion ratio and the conversion was stopped by addition of ethanol at >50% (v/v). The isomerization was significantly affected pH (2-10) and the boiling medium at pH 5 was minimum for the conversion. Therefore, it was suggested that xanthohumol was relatively difficult to convert to isoxanthohumol in wort (pH 5-5.5) during boiling.
Assuntos
Cerveja/análise , Cromatografia Líquida de Alta Pressão/métodos , Flavanonas/isolamento & purificação , Xantonas/isolamento & purificação , Flavanonas/análise , Flavanonas/química , Humulus/química , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Estereoisomerismo , Xantonas/análise , Xantonas/químicaRESUMO
Plastid division is controlled by numerous nuclear genes. Arabidopsis thaliana CRUMPLED LEAF (AtCRL) is a plastid division-related gene, and the crl mutant exhibits a dwarf phenotype with abnormal cell division and a significant reduction in plastid numbers. However, the function of AtCRL is not fully understood. Here, we identified and characterized two AtCRL homologs, PpCRL1 and PpCRL2, in the moss Physcomitrella patens. PpCRL1 and PpCRL2 shared 77% amino acid identity with each other and 47% identity with AtCRL. Single PpCRL1 or -2 gene knockout (KO) mutants could not be distinguished from the wild-type mosses, but PpCRL1 and -2 double KO mutants displayed growth retardation of protonemata and gametophores and harbored approximately 10 large chloroplasts per cell. This indicates that PpCRL1 and PpCRL2 have redundant functions in chloroplast division and plant growth. Unlike the A. thaliana crl mutants, however, the PpCRL double KO mutants did not display abnormal orientation of the cell division plane. Complementation experiments showed that AtCRL partially rescued the defects in chloroplast size and number of the PpCRL double KO mutant. This suggests that PpCRL has a similar, but not identical, function to AtCRL. Time-lapse microscopic observation of the double PpCRL KO mutants revealed that some dumbbell-shaped chloroplasts failed to complete division at the late stage of plastid division; enlarged chloroplasts were thus generated. This strongly suggests that PpCRLs are involved in the complete separation of dividing chloroplasts.
Assuntos
Bryopsida/genética , Proteínas de Plantas/metabolismo , Plastídeos/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bryopsida/crescimento & desenvolvimento , Bryopsida/fisiologia , Divisão Celular , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga , Dados de Sequência Molecular , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/fisiologia , Plastídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Imagem com Lapso de TempoRESUMO
The human general transcription factor, TFIIE, consists of two subunits, alpha and beta. Structural analyses indicated the presence of a forkhead motif within the central region of TFIIEbeta. This motif was essential for transcription and possessed a double-stranded DNA-binding activity. Protein-DNA photo-cross-linking studies indicated that TFIIEbeta binds within the promoter region, adjacent to the transcription initiation site where promoter melting begins at transcription initiation. Furthermore, neither TFIIE nor the other general transcription factor TFIIH, were required for basal transcription of adenovirus major late promoter artificially pre-melted at the initiation site. These data suggest a model in which TFIIE binds to a position adjacent to the initiation site via the forkhead domain, enabling TFIIH to begin opening the promoter. Here, we used systematic point mutations to further investigate the functional roles of this domain. The mutant proteins were expressed in bacteria, purified and used to examine transcription of two different forms of template, phosphorylation of the C-terminal domain of RNA polymerase II, as well as dsDNA-binding. Taken together, our results strongly demonstrated that the primary function of the forkhead region is dsDNA-binding in transcription. In addition, we identified three positively charged lysine residues which play a key role in this function.
