Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation.
Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation. In total, 16 samples were analyzed of which 4 were control (MED) samples, 4 samples of p31-43 treatment, 4 samples of PT treatmetn and 4 samples of PT-G treatment
Project description:Whole-cell lysates were digested with trypsin, fractionated by reverse phase chromatography into 24 fractions and analyzed by liquid chromatography - tandem mass spectrometry
Project description:We have comprehensively profiled the time-series changes in gene expression dependent on enzymatic treatment using trypsin, and attempted to elucidate the global changes suppressed by treatment with cold active protease in a low-temperature environment.
Project description:A DNA-peptide complex that is soluble in 0.2m-sodium chloride can be prepared by trypsin digestion of calf thymus nucleoprotein. The trypsin-digested nucleoprotein molecule contains about 70% of DNA and 30% of peptides by weight, and consists of one DNA molecule associated with arginine-rich peptides. A series of trypsin-digested nucleoprotein preparations differing only in molecular weight were prepared by blending. The intrinsic viscosity and average sedimentation coefficient were determined for each of these preparations. Then the DNA was isolated from each preparation and the hydrodynamic measurements were repeated on the DNA. From a comparison of these results it was concluded that the presence of the complex-forming peptides causes a large decrease in intrinsic viscosity of the DNA and an increase in sedimentation coefficient. In addition, the hydrodynamic data indicate that the DNA-peptide complex behaves like a coil in solution but is more compact than the same length of DNA. The ;melting' profiles, streptomycin precipitation curves and maximum viscosities obtained with ethidium bromide binding for the trypsin-digested nucleoprotein are similar to those of purified DNA, and markedly different from those of undigested nucleoprotein. These findings suggest that the peptides are not strongly associated with the DNA, and that secondary valency forces are involved in the binding.
Project description:The level of trypsin-2 has been shown to correlate with the malignancy and metastatic potential of many cancer. We used microarrays to detail how tryspin-2 overexpression changes the gene profile of human tongue squamous cell carcinoma cell line (HSC-3). We compared trypsin-2 overexpressing HSC-3 line to HSC-3 which was transfected with control vector and because of that did not expressed trypsin-2 at all.
Project description:The level of trypsin-2 has been shown to correlate with the malignancy and metastatic potential of many cancer. We used microarrays to detail how tryspin-2 overexpression changes the gene profile of human tongue squamous cell carcinoma cell line (HSC-3).
Project description:Human embryonic kidney cell (HEK293) were treated with PAR2 peptide agonist 2f-LIGRLO-NH2 (1.5h, 3h, 6h and 12h) or trypsin (6h). Comparison of genes similarly regulated by both treatments allowed better characterization of PAR2 induced response as both agonists had been reported as non-specific for PAR2.