RESUMO
The enzyme tissue transglutaminase (tTG) has been recently identified to represent a highly sensitive and specific target of autoantibodies in coeliac disease. To characterize autoantigenic epitopes, we generated novel tTG deletion mutants by polymerase chain reaction, produced radiolabelled fragments by in vitro transcription/translation, immunoprecipitated the mutants using sera from patients with coeliac disease, and related the binding data with putative structural and functional domains of human tTG. We show that tTG antibody positive sera display a heterogeneous autoantibody response covering distinct regions of the molecule. The N-terminal and C-terminal third of tTG, comprising amino acid (aa) 1-281 and aa 473-687, harbour the dominant epitopes (67.4% and 69.4% positive), whereas the catalytic region is of minor antigenicity (22.5% positive). Autoantibodies directed to one, two and three domains were observed in 36.7%, 28.6% and 22.4% of patients, respectively. Comparative analysis revealed the presence of strictly conformational epitopes which were dependent on the N-terminus (aa 1-12) or the intact beta-barrel domains in the C-terminus (aa 473-497, aa 649-687). In conclusion, we here demonstrate for the first time that the humoral autoimmunity is directed against distinct functional tTG domains. The spectrum of autoantibodies indicates that the native folded protein may be the target of autoantibodies.
Assuntos
Especificidade de Anticorpos , Autoanticorpos/imunologia , Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/imunologia , Transglutaminases/imunologia , Adolescente , Adulto , Idoso , Autoantígenos/química , Autoantígenos/genética , Criança , Pré-Escolar , Epitopos/imunologia , Feminino , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Glutamina gama-Glutamiltransferase , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Transglutaminases/química , Transglutaminases/genéticaRESUMO
After oral administration of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-piperazine-1-ylquinoline++ +-3-carboxylic acid (ciprofloxacin, Bay o 9867; designated trademark: Ciprobay) four metabolites M1-M4 were isolated from human urine by Craig counter current distribution and semipreparative high-performance liquid chromatography. Their molecular structures were elucidated by nuclear magnetic resonance and mass spectrometry and confirmed by comparing their spectra with those of authentic synthetic reference compounds.
Assuntos
Ciprofloxacina/metabolismo , Administração Oral , Adulto , Cromatografia Líquida de Alta Pressão , Ciprofloxacina/administração & dosagem , Ciprofloxacina/isolamento & purificação , Ciprofloxacina/urina , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Pessoa de Meia-IdadeRESUMO
Etofenamate [2-(2-hydroxyethoxy)ethyl-N-(alpha, alpha, alpha-trifluoro-m-tolyl)-anthranilate] was administered to dogs by the oral route. Minor amounts of etofenamate (Eto) and its glucuronide were found in urine and feces. The main portion of metabolites was eliminated as flufenamic acid (Flu) and hydroxy derivatives of Eto and Flu. Furthermore, a highly lipophilic fraction was isolated (extraction and TLC) and further separated into several compounds (HPLC, GLC). These metabolites were identified as Eto oleate, palmitate, linoleate, stearate, palmitoleate, myristate, and laurate by NMR and MS. The structures were confirmed by comparison with authentic material. The conjugation of etofenamate with fatty acids is an example of a new route of drug metabolism.
