RESUMO
4-Nonylphenol is a widely diffused and stable environmental contaminant, originating from the degradation of alkyl phenol ethoxylates, common surfactants employed in several industrial applications. Due to its hydrophobic nature, 4-nonylphenol can easily accumulate in living organisms, including humans, where it displays a wide range of toxic effects. Since the gastrointestinal tract represents the main route by which 4-nonylphenol enters the body, the intestine may be one of the first organs to be damaged by chronic exposure to this pollutant through the diet. In the present study, we investigated the effects of 4-nonylphenol on a human intestinal epithelial cell line (Caco-2 cells). We demonstrated that 4-nonylphenol was cytotoxic to cells, as revealed by a decrease of the cell number and the decrement of mitochondrial functionality after 24 h of treatment. 4-Nonylphenol also reduced the number of cells entering into S-phase and interfered with epidermal growth factor signalling, with consequent negative effects on cell survival. In addition, 4-nonylphenol induced apoptosis, involving the activation of caspase-3, and triggered an endoplasmic reticulum-stress response, as revealed by over-expression of GRP78 (78 kDa glucose-regulated protein) and activation of XBP1 (X-box binding protein-1). Together, these findings support the hypothesis that prolonged exposure to 4-nonylphenol through the diet may lead to local damage at the level of intestinal mucosa, with potentially negative consequences for intestinal homeostasis and functionality.
Assuntos
Mucosa Intestinal/efeitos dos fármacos , Fenóis/toxicidade , Apoptose/efeitos dos fármacos , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Anti-transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti-transglutaminase antibodies. The aim was to measure positive anti-transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti-transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty-two children suffering from infectious diseases were enrolled prospectively along with seven biopsy-proven coeliacs. Serum samples were tested for anti-transglutaminase antibodies and anti-endomysium antibodies; positive samples were tested for coeliac-related human leucocyte antigen (HLA)-DQ2/8 and anti-viral antibodies. Purified anti-transglutaminase antibodies from the two study groups were tested for urea-dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell-cycle in Caco-2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti-transglutaminase, one of whom also tested positive for anti-endomysium antibodies. This patient was positive for HLA-DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA-DQ8. Levels of anti-transglutaminase returned to normal in all subjects, despite a gluten-containing diet. Purified anti-transglutaminase of the two study groups induced actin rearrangements and cell-cycle progression. During an infectious disease, anti-transglutaminase antibodies can be produced temporarily and independently of gluten. The infection-triggered anti-transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in-vivo potential for damage.
Assuntos
Autoanticorpos/imunologia , Doença Celíaca/imunologia , Doenças Transmissíveis/imunologia , Transglutaminases/imunologia , Actinas/metabolismo , Adolescente , Anticorpos/farmacologia , Autoanticorpos/sangue , Células CACO-2 , Doença Celíaca/sangue , Doença Celíaca/diagnóstico , Ciclo Celular/efeitos dos fármacos , Criança , Pré-Escolar , Doenças Transmissíveis/sangue , Doenças Transmissíveis/diagnóstico , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Microscopia de Fluorescência , Estudos ProspectivosRESUMO
Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in 'toxic' and 'immunogenic' classes, which elicit an innate immune response and an HLA-mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31-43 (P31-43) and the 56-68 (P56-68) A-gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane-mimetic medium. We show that P31-43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56-68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD.
Assuntos
Membrana Celular/metabolismo , Gliadina , Fragmentos de Peptídeos , Doença Celíaca/metabolismo , Membrana Celular/química , Corantes/química , Detergentes/química , Gliadina/química , Gliadina/imunologia , Gliadina/toxicidade , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Micelas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Dodecilsulfato de Sódio/químicaRESUMO
Celiac disease (CD) is an autoimmune pathology of the small intestine triggered, in genetically predisposed patients, by the exposition to gliadin, a flour protein, thus evoking local immune reactions and mucosal atrophy. The discovery that type 2 transglutaminase (TG2) is the main, if not the sole, target of the endomysium CD-specific autoantibodies assigned to this enzyme a master regulator role of CD. Two separated events, both based on the finding that gliadin is able to act as a TG2 substrate, have been described to indicate that TG2 is involved in both the humoral and cellular immune responses. In this paper we review the novel insights on the localization and enzymatic activity of TG2 in the small intestinal mucosa. Moreover, we report on the capability of gliadin and its peptides to act as TG2 substrates.
Assuntos
Doença Celíaca/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Gliadina/metabolismo , Transglutaminases/metabolismo , Glutens/química , Glutens/genética , Glutens/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Intestino Delgado/anatomia & histologia , Intestino Delgado/metabolismo , Isoenzimas/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteína 2 Glutamina gama-GlutamiltransferaseRESUMO
BACKGROUND AND AIMS: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity. METHODS: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord. RESULTS: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl(2) caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies. CONCLUSIONS: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.
Assuntos
Anticorpos Monoclonais/farmacologia , Doença Celíaca/enzimologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/imunologia , Transglutaminases/imunologia , Animais , Cloreto de Cálcio/farmacologia , Doença Celíaca/imunologia , Linhagem Celular , Células Cultivadas , Cães , Proteínas de Ligação ao GTP/análise , Humanos , Imunoglobulina A/farmacologia , Imunoglobulina G/farmacologia , Imuno-Histoquímica/métodos , Camundongos , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/farmacologia , Transglutaminases/análise , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismo , Cordão Umbilical/enzimologiaRESUMO
The in vitro metabolism of transglutaminase-synthesized substance P analogs has been characterized comparing their stability to that of the parent peptide. The major metabolites have been purified and their structures elucidated by mass spectrometry. Our results demonstrated that gln5 spermidine and spermine analogs of substance P possess an enhanced resistance to the action of proteases. Moreover spermine, a large size hydrophilic compound, specifically prevented the hydrolysis at Phe7-Phe8 bond.
Assuntos
Aminas/metabolismo , Substância P/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Hidrólise , Dados de Sequência Molecular , Peptidil Dipeptidase A/metabolismo , Ratos , Substância P/análogos & derivadosRESUMO
The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at approximately 1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources.