AIM： To evaluate the effects of gliadin on the oxidative environment in the “in vivo-like”model of a three-dimensional cell culture system. METHODS： LoVo cell line （intestinal adenocarcinoma） multicellular spher...AIM： To evaluate the effects of gliadin on the oxidative environment in the “in vivo-like”model of a three-dimensional cell culture system. METHODS： LoVo cell line （intestinal adenocarcinoma） multicellular spheroids were treated with digested gliadin （with albumin used as a control）. Spheroid volumes, cell viability and morphology, lactate dehydrogenase （LDH） release, content of reduced glutathione （GSH） and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student＇s t-test （P〈0.05）. was considered statistically significant. RESULTS： Gliadin reduced cell viability （from 20% to 60%） and led to morphological alterations characterized by apoptotic findings and cytoskeletal injuries. LDH activity increased. The content of GSH reduced （-20% vscontrols）, and activity of GSH-related enzymes was significantly inhibited. CONCLUSION： Gliadin treatment induces an imbalance in the antioxidative mechanism of cells cultured by the three-dimensional technique. This alteration may explain cell damage directly caused by gliadin and the subsequentmorphological abnormalities.展开更多
AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to ...AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.展开更多
The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on ...The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.展开更多
基金Supported by the San Paolo Foundation grant to "Centro per lo Studio della Celiachia"
文摘AIM： To evaluate the effects of gliadin on the oxidative environment in the “in vivo-like”model of a three-dimensional cell culture system. METHODS： LoVo cell line （intestinal adenocarcinoma） multicellular spheroids were treated with digested gliadin （with albumin used as a control）. Spheroid volumes, cell viability and morphology, lactate dehydrogenase （LDH） release, content of reduced glutathione （GSH） and activity of GSH-related enzymes were examined. The data were statistically analyzed using the Student＇s t-test （P〈0.05）. was considered statistically significant. RESULTS： Gliadin reduced cell viability （from 20% to 60%） and led to morphological alterations characterized by apoptotic findings and cytoskeletal injuries. LDH activity increased. The content of GSH reduced （-20% vscontrols）, and activity of GSH-related enzymes was significantly inhibited. CONCLUSION： Gliadin treatment induces an imbalance in the antioxidative mechanism of cells cultured by the three-dimensional technique. This alteration may explain cell damage directly caused by gliadin and the subsequentmorphological abnormalities.
基金Supported by the "Fondazione San Paolo" grant to "Centro perlo Studio della Celiachia"
文摘AIM： To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns.METHODS： We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure （by means of light microscopy and scanning electron microscopy）, and the effect of gliadin on actin （phalloidin fluorescence） and the tight-junction protein occludin and zonula occluden-1.RESULTS： The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gUadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system.CONCLUSION： Our data obtained from an ＂in vivolike＂ polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.
文摘The occurrence of twin-arginine motifs （-R-R-） in the amino acid sequences of animal pro-proteins frequently defines the cleavage site（s） for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the pres- ence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry anal- yses, These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-（2-AminoEthyl）Benzene-Sulphonyl Fluoride hy- drochloride （AEBSF）, leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans- EpoxysuccinyI-L-leucylamido（4-guanidino）butane （E64）, and ethylenediaminetetraacetic acid （EDTA）, thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.
