RESUMO
Eumelanin photoprotects pigmented tissues from ultraviolet (UV) damage. However, UVA-induced tanning seems to result from the photooxidation of preexisting melanin and does not contribute to photoprotection. We investigated the mechanism of UVA-induced degradation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-melanin taking advantage of its solubility in a neutral buffer and using a differential spectrophotometric method to detect subtle changes in its structure. Our methodology is suitable for examining the effects of various agents that interact with reactive oxygen species (ROS) to determine how ROS is involved in the UVA-induced oxidative modifications. The results show that UVA radiation induces the oxidation of DHICA to indole-5,6-quinone-2-carboxylic acid in eumelanin, which is then cleaved to form a photodegraded, pyrrolic moiety and finally to form free pyrrole-2,3,5-tricarboxylic acid. The possible involvement of superoxide radical and singlet oxygen in the oxidation was suggested. The generation and quenching of singlet oxygen by DHICA-melanin was confirmed by direct measurements of singlet oxygen phosphorescence.
Assuntos
Indóis/química , Melaninas/química , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria/métodos , Raios Ultravioleta , Animais , Ácido Ascórbico/farmacologia , Boroidretos/farmacologia , Bovinos , Peróxido de Hidrogênio/farmacologia , Indóis/efeitos da radiação , Luminescência , Melaninas/efeitos da radiação , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Oxigênio Singlete/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Fatores de TempoRESUMO
Human macrophage dectin-1, a type II transmembrane ß-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag in a baculovirus-silkworm expression system and assayed for binding activity. Recombinant dectin-1 specifically bound to some ß-glucans, and the neck domain and N-linked oligosaccharide chains of human dectin-1 did not affect the ligand binding activity and specificity of the receptor.
Assuntos
Baculoviridae/genética , Bombyx/genética , Engenharia Genética/métodos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , beta-Glucanas/metabolismo , Animais , Linhagem Celular , Expressão Gênica , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Human macrophage dectin-1, a type II transmembrane beta-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and assayed for binding specificity. Recombinant dectin-1 specifically bound to some beta-glucans, but not to other carbohydrates. The beta-glucan binding of recombinant dectin-1 was inhibited by laminarin, a soluble beta-glucan, and by laminarioligosaccharides, but not by other carbohydrates. These results suggest that recombinant human dectin-1 can be used as a useful probe in identifying ligands in humans and tonic foods due to its strict binding specificity.