Assuntos
Citoproteção/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Oftalmopatia de Graves/tratamento farmacológico , Peróxido de Hidrogênio/toxicidade , Órbita/efeitos dos fármacos , Selênio/farmacologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Idoso , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Feminino , Fibroblastos/patologia , Oftalmopatia de Graves/patologia , Oftalmopatia de Graves/prevenção & controle , Humanos , Masculino , Pessoa de Meia-Idade , Órbita/patologia , Cultura Primária de Células , Selênio/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
DigesTip is a new device for in-solution protein digestion, based on a patent pending technology, able to immobilize enzymes (trypsin, in this case) on a solid surface, keeping their activity preserved. DigesTip is a standard pipette tip, usable both by human and by robots. Its main performances are: very short digestion time (1 min) and usability with low protein sample concentrations (5 microg/mL). DigesTip obtains a clear signal in MS measurements and its usage rules out several preparative steps.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodos , Biotecnologia/métodos , Desenho de Equipamento , Humanos , Concentração de Íons de Hidrogênio , Indústrias , Cinética , Espectrometria de Massas/instrumentação , Peptídeos/química , Proteínas/química , Albumina Sérica/química , Temperatura , Fatores de Tempo , Tripsina/químicaRESUMO
The goal of this study was to map an epitope on the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) at its C terminus, a region whose integrity is fundamental in maintaining the normal function of this molecule. Residues including the fourth alpha-helix (D, 103-116) were analyzed for their role in the interaction with antibodies (Abs) raised against the protein. Five peptides homologous to different segments of the C terminus of hGM-CSF were synthesized. Peptide-(102-121) included the same residues of the alpha-helix D and the next five amino acids toward the C terminus; peptide-[E108A]-(102-121) introduced the mutation E108A in order to verify the role of acidic residues; peptide-[C96A](93-110) encompassed the beta-sheet 2 and half of the alpha-helix D; peptide-[C121A]-(110-127) included the second half of the alpha-helix D and the C terminus of hGMCSF; peptide-(13-31)-Gly-Pro-Gly-(103-116) included both the alpha-helices A and D connected by the tripeptide Gly-Pro-Gly, which allows the original antiparallel orientation of the two alpha-helices to be maintained. Both anti-protein and anti-peptide-(102-121) antibodies, capable of neutralizing the stimulatory activity of hGMCSF in the bone marrow colony-forming assays, recognized a specific epitope in the C terminus of hGM-CSF. Molecular modeling estimated the surface accessibility of hGM-CSF and the stability of the synthetic peptides in aqueous solution. Altogether, our results showed that the immunogenic region includes part of the alpha-helix D and the residues 116-120, which are external to this helix and particularly exposed on the protein surface, confirming the feasible participation of this region in antibody binding.
