Project description:Background: Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Dietary fibres have been descirbed to be beneficial for intestinal health. Therefore, in this study we explored the applicability of an in vitro model, namely human intestinal Caco-2 cells, to study the effect of dietary fibres on intestinal health. Transcriptomics was applied to obtain more insight into their mode of actions in the intestinal cells. Methods: Caco-2 cells were stimulated with 500 ug/ml dietary fibres and the maximal observed LPS contamination to serve as background control for 6 hours, total RNA was extracted and Affymterix Human Gene 1.1 ST arrays were used to analyze the gene expression profiles. To identify dietary fibre induced gene expression profiles in dietary fibre gene responses were compared to medium samples. Furthermore, to analyse differentiatlly affected pathways Ingenuite Pathway Analysis was employed. Results: Pathway analysis revealed a distinct separation between the dietary fibres. GOS and beta-glucan oat medium viscosity affected transcription of a lower amount of genes (gene cut-off p<0.05) and gen transcription changes suggest an increase in vesicle transport and altered cholesterol regulation. On the other hand, the other dietary fibres differentially regulated a larger numbers of genes (gene cut-off p<0.05) and all appeared related to immune responses. We observed an increase in intracellular and extracellular anti-bacterial pathways and production of cytokines specifically aimed at communication with the adaptive immune system. Conclusion: GOS and beta-glucan oat medium viscosity appeared to induce intestinal epithelial communication with the body, whereas the other dietary fibres appeared recognized as PAMP and induce epithelial cells to interact with the immune system.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Background: Macrophages represent an important part of the immune system in the intestine and are crucial for maintaining homeostasis. As part of research investigating the effect of dietary fibres on the intestinal immune barrier THP-1 macrophages were used as model system. Methods: THP-1 monocytes were stimulated for 48 hours with 100 ng/ml PMA and 48 hours rested in medium. Subsequently, they were stimulated with 500 ug/ml dietary fibres and the maximal observed LPS contamination to serve as background control. After 6 hours, total RNA was extracted and Affymterix Human Gene 1.1 ST arrays were used to analyze the gene expression profiles. To identify dietary fibre induced gene expression profiles in dietary fibre gene responses were compared to medium samples. Furthermore, to analyse differentiatlly affected pathways Ingenuite Pathway Analysis was employed. Results: There was a clear difference in significantly differentially expressed genes (gene cut-off p<0.05) with beta-glucan oat medium viscosity and GOS changing transcription of a relative small amount of genes and Sugar beet pectin and Resistant starch a relative large amount of genes. These latter two were also the only dietary fibres to demonstrate an increase in Fc-receptor-related pathway activation. Alternatively, beta-glucan oat medium viscosity and GOS were the only dietary fibres to activate pathways related to cellular movement and the only two to not activate the Ahr-signaling pathway (p<0.05). Conclusion: our data indicate that the in vitro THP-1 macrophage model can be used to differentiate in immunomodulatory potential of dietary fibres and provide hypotheses for functional differentiation.
Project description:Increasing the consumption of dietary fibre has been proposed to alleviate the progression of non-communicable diseases such as obesity, type 2 diabetes and cardiovascular disease, yet the effect of dietary fibre on host physiology remains unclear. In this study, we performed a multiple diet feeding study in C57BL/6J mice to compare high fat and high fat modified with dietary fibre diets on host physiology and gut homeostasis by combining proteomic, metagenomic, metabolomic and glycomic techniques with correlation network analysis. We observed significant changes in physiology, liver proteome, gut microbiota and SCFA production in response to high fat diet. Dietary fibre modification did not reverse these changes but was associated with specific changes in the gut microbiota, liver proteome, SCFA production and colonic mucin glycosylation. Furthermore, correlation network analysis identified gut bacterial-glycan associations.