Products formed during fermentation of the prebiotic inulin with humangut flora enhance expression of biotransformation genes in human primarycolon cells

Inulin-type fructans are fermented by gut bacteria to yield SCFA, including butyrate which is trophic for colonocytes and induces glutathioneS-transferases (GST) that detoxify carcinogens. Since little is known on similar effects by complex fermentation samples, we studied related products in non-transformed human colonocytes. Inulin enriched with oligofructose (1 : 1, Synergy1) was fermented with human gut flora. SCFA were quantified and a SCFA mixture was prepared accordingly. Colonocytes were incubated (4–12 h) with the Synergy1 fermentation supernatant (SFS), faeces control, a mixture of the three major SCFA (each 0–15 %, v/v) or butyrate (0–50 mm). Metabolic activity was determined to assess trophic effects and cytotoxicity. Expression of ninety-six genes related to biotransformation was studied using cDNA macroarrays. Results on modulated GST were reassessed with real-time PCR and GST activity was measured. Fermentation of inulin resulted in 2–3-fold increases of SCFA. The samples were non-cytotoxic. SFS increased metabolic activity, pointing to trophic effects. The samples modulated gene expression with different response patterns. Key results were thatGSTM2(2·0-fold) andGSTM5(2·2-fold) were enhanced by SFS, whereas the SCFA mixture reduced expression. The faeces control enhancedGSTA4(2·0-fold), but reducedGSTM2(0·2-fold) andGSTM5(0·2-fold). Real-time qPCR confirmed the induction ofGSTM2andGSTM5by SFS and ofGSTA4andGSTT2by butyrate. Activity of GST was not modulated. High concentrations of fermentation products were well tolerated by primary colonocytes, pointing to trophic effects. The induction of GST by the SFS may protect the cells from carcinogenic compounds.

Sporangium-Specific Gene Expression in the Oomycete Phytopathogen Phytophthora infestans

ABSTRACT The oomycete genus Phytophthora includes many of the world's most destructive plant pathogens, which are generally disseminated by asexual sporangia. To identify factors relevant to the biology of these propagules, genes induced in sporangia of the potato late blight pathogen Phytophthora infestans were isolated using cDNA macroarrays. Of ∼1,900 genes known to be expressed in sporangia, 61 were up-regulated >5-fold in sporangia versus hyphae based on the arrays, including 17 that were induced> 100-fold. A subset were also activated by starvation and in a nonsporulating mutant. mRNAs of some genes declined in abundance after germination, while others persisted through the germinated zoospore cyst stage. Functions were predicted for about three-quarters of the genes, including potential regulators (protein kinases and phosphatases, transcription factors, and G-protein subunits), transporters, and metabolic enzymes. Predominant among the last were several dehydrogenases, especially a highly expressed sorbitol dehydrogenase that accounted for 3% of the mRNA. Sorbitol dehydrogenase activity also rose during sporulation and several stress treatments, paralleling the expression of the gene. Another interesting metabolic enzyme resembled creatine kinases, which previously were reported only in animals and trypanosomes. These results provide insight into the transcriptional and cellular processes occurring in sporangia and identify potential targets for crop protection strategies.

Acute Hyperglycaemia Induces Changes in the Transcription Levels of 4 Major Genes in Human Endothelial Cells: Macroarrays-based Expression Analysis

SummaryHyperglycaemia, in insulin-dependent or independent diabetes mellitus, promotes endothelial cell (EC) dysfunction and is a major factor in the development of macro- or microvascular diseases. The mechanisms and the disease-related genes in vascular diseases resulting from hyperglycaemia are poorly understood. Macroarrays, bearing a total of 588 cDNA known genes, were used to analyze HUVEC gene transcription subjected to 25 or 5-mM glucose for 24 h. Isolated mRNA derived from treated first passage HUVEC were reverse transcribed, 32P labeled, and hybridized to the cDNA macroarrays. Results show that acute hyperglycaemia induces an up-regulation of seven major genes, four of which were not previously reported in the literature. Northern blot analyses, performed on these 4 genes, confirm macroarrays results for αv, β4, c-myc, and MUC18. Moreover, time course analysis (0, 2, 4, 8, 12, 16, 24 h) of αv, β4, c-myc, and MUC18 mRNA expression, observed by northern blot assays, showed a peak at time points situated between 2 to 8 h. The 3 other genes (ICAM-1, β1, and IL-8), were shown by others to be significantly upregulated after glucose stimulation. Furthermore, ELISA assays performed on the supernatant of HUVEC culture medium showed a significant increase of IL-8 for cells treated with 25-mM compared to 5-mM glucose. Identified genes, upregulated in endothelial cells as a result of acute hyperglycaemia, may serve as therapeutic or diagnostic targets in vascular lesions present in diabetic patients. These results also demonstrate the use of cDNA macroarrays as an effective approach in identifying genes implicated in a diseased cell.