2, 4-Diamino-6- hydroxy pyrimidine inhibits NSAIDs induced nitrosyl-complex EPR signals and ulcer in rat jejunum

BACKGROUND: It has been suggested that one aspect of non-steroidal anti-inflammatory drugs induced intestinal damage is due to either uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport. We investigated the latter possibility using electron paramagnetic resonance spectroscopy. RESULTS: Electron paramagnetic studies of NSAIDS on sub-mitochondrial particles revealed that indomethacin, but not with nabumetone, bound to a site near to Complex I and ubiquinone to generate a radical species. Normal rats exhibited prominent [3Fe-4S]ox signals (g ~ 2.01) at 20 K. One hour after indomethacin there was a prominent, intense and broad absorption pattern at (g ~2.07) suggesting, appearance of radical species overlapping [3Fe-4S]ox and was unaffected by pretreatment with 2,4 diamino -6-hydroxy pyrimidine. At 24 hrs, when macroscopic ulcers were seen, there was a new signal due to a nitric oxide radical (NO•). In contrast, nabumetone and 2,4 diamino-6-hydroxy pyrimidine pre-treated animals receiving indomethacin exhibited electron paramagnetic resonance spectra identical to those of controls at 24 hrs and neither was associated with small intestinal ulcers. Indomethacin and 2,4 diamino hydroxy pyrimidine pre-treated rats, but not nabumetone, had increased intestinal permeability. CONCLUSION: The results suggest that the in vivo effects of indomethacin modulate the mitochondrial respiratory chain directly at 1 h and 24 h through formation of nitric oxide. NO• appears to play an important role in the late pathogenic stages of NSAID enteropathy and may be the site for targeted treatment to reduce their toxicity

A Factor Graph Nested Effects Model To Identify Networks from Genetic Perturbations

Complex phenotypes such as the transformation of a normal population of cells into cancerous tissue result from a series of molecular triggers gone awry. We describe a method that searches for a genetic network consistent with expression changes observed under the knock-down of a set of genes that share a common role in the cell, such as a disease phenotype. The method extends the Nested Effects Model of Markowetz et al. (2005) by using a probabilistic factor graph to search for a network representing interactions among these silenced genes. The method also expands the network by attaching new genes at specific downstream points, providing candidates for subsequent perturbations to further characterize the pathway. We investigated an extension provided by the factor graph approach in which the model distinguishes between inhibitory and stimulatory interactions. We found that the extension yielded significant improvements in recovering the structure of simulated and Saccharomyces cerevisae networks. We applied the approach to discover a signaling network among genes involved in a human colon cancer cell invasiveness pathway. The method predicts several genes with new roles in the invasiveness process. We knocked down two genes identified by our approach and found that both knock-downs produce loss of invasive potential in a colon cancer cell line. Nested effects models may be a powerful tool for inferring regulatory connections and genes that operate in normal and disease-related processes

p16INK4a Gene Promoter Hypermethylation in Mucosa as a Prognostic Factor for Patients with Colorectal Cancer

Low gene expression of folylpolyglutamate synthase (FPGS) in colorectal mucosa correlates with low folate levels and poor survival of colorectal cancer (CRC) patients. Because gene-specific hypermethylation is affected by the folate level, the hypermethylation status in mucosa may also be linked to clinical outcome of CRC patients. The tumor suppressor gene p16INK4a (p16) regulates the cell cycle and angiogenic switch. In human neoplastic tissues, the main mechanism of p16 inactivation is promoter methylation. The aim of the study was to determine whether hypermethylation of the p16 promoter could be detected in mucosa of CRC patients (n = 181) and to analyze if hypermethylation was related to survival. The relation between p16 hypermethylation and expression of FPGS and two other folate-associated genes, reduced folate carrier 1 (RFC-1), and thymidylate synthase (TS), was analyzed (n = 63). The results showed that p16 was hypermethylated in 65 (36%) of the mucosa samples and that hypermethylation was age-related (P = 0.029). After adjustment for known risk factors, Cox regression analysis showed that Dukes’ A-C patients with p16 hypermethylation in mucosa had an increased risk of cancer-related death (hazard ratio = 2.9, P = 0.007) and shorter disease-free survival (hazard ratio = 2.5, P = 0.015) compared with patients with no p16 hypermethylation. RFC-1 and FPGS gene expression levels were significantly correlated in patients lacking p16 hypermethylation in mucosa (P = 0.0003), but not at all correlated in patients having hypermethylation in mucosa (P = 1.0). In conclusion, p16 hypermethylation in mucosa of CRC patients was identified as an independent prognostic parameter for cancer-specific survival as well as an independent predictor of DFS. The results suggest that there might be a connection between folate-associated gene expression and p16 methylation status