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
The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMAIII) and compared with that of trivalent arsenite (iAsIII) and monomethylarsonous acid (MMAIII). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMAIII, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAsIII or MMAIII. The generation of reactive oxygen species (ROS) after DMAIII exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMAIII. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMAIII-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAsIII and MMAIII. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMAIII-induced cytotoxicity.
Assuntos
Arsenitos/toxicidade , Ácido Cacodílico/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Compostos Organometálicos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fosforilação/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/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.
