RESUMO
Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5alpha-cholestan-3beta-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO(3), pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.
Assuntos
Ésteres do Colesterol/síntese química , Hidroxicolesteróis/síntese química , Cetocolesteróis/síntese química , Ésteres do Colesterol/química , Óxido de Etileno/química , Hidrocarbonetos Fluorados/química , Hidroxicolesteróis/química , Cetocolesteróis/químicaRESUMO
Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP(2). To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP(2) can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP(2) in the cytoplasm of muscle cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cofilina 1/metabolismo , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Ligação Proteica/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Sequência de Aminoácidos/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células Cultivadas , Embrião de Galinha , Cofilina 1/farmacologia , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Células Musculares/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Miofibrilas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A direct method for the separation and quantification of a series of bile acid acyl glycosides using high-performance liquid chromatography coupled to an evaporative light scattering detector (HPLC-ELSD) is described. Complete separation of each of 15 bile acid acyl 24-alpha-glucosides and their 24-beta-anomers and 24-beta-galactosides was achieved by the stepwise gradient elution mode on a C18 column using a mixture of acetonitrile-methanol (8:2, v/v) and 1% aqueous acetic acid as the mobile phase. 24-beta-Galactosides were always eluted faster than the corresponding 24-beta-glucosides, which eluted after the corresponding 24-alpha-anomers. Calibration curves of different 24-beta-galactosides were linear over a range of 0.2-40 nmol of injected amount and the detection limits (S/N > 3) were from 0.08 to 0.1 nmol. The present HPLC-ELSD method may provide an insight into the separation and quantification of the biologically interesting neutral bile acids.
Assuntos
Ácidos e Sais Biliares/química , Cromatografia Líquida de Alta Pressão/métodos , Glicosídeos/análise , Espalhamento de Radiação , Ácidos e Sais Biliares/normas , Calibragem , Glicosídeos/química , Glicosídeos/isolamento & purificação , Estrutura Molecular , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A methanol extract of the pericarps of Illicium minwanense afforded seven new seco-prezizaane-type sesquiterpenes (2-8) and a new abietane-type diterpene (9), together with six previously known compounds (1 and 10-14). The structures of the new compounds, (1S)- and (1R)-minwanenone (2 and 3), 1alpha-hydroxy-6-deoxypseudoanisatin (4), (2S)-hydroxy-6-deoxypseudoanisatin (5), 3-oxopseudoanisatin (6), (3S,6R)-4,7-epoxy-6-deoxypseudoanisatin (7), 7-O-methylpseudomajucin (8), and (+)-8,11,13,15-abietatetraene (9), were elucidated by spectroscopic data analysis and chemical transformations. The absolute configurations of 1 and 5 were established by X-ray crystallographic analysis of their p-bromobenzoyl derivatives.
