A canine model for the study of gastric secretion and emptying after a meal

Our aim was to develop a chronic canine model that would serve for the simultaneous measurements of postprandial gastric secretion and emptying by a double-marker dilution technique without artificially interfering with intragastric pH. A constant duodenal perfusion of a nonabsorbable marker allowed determination of luminal flow and total recovery of a second marker ingested with a meal. By calculating the amount of meal marker remaining in the stomach and its dilution (by repeat gastric sampling), we could determine the volume of gastric contents. Acid concentration in gastric samples was measured by titration ex vivo, and gastric acid content was calculated by multiplying the volume of gastric contents by titratable acidity. In this model, net acid gain at each sampling interval indicates acid output. Acid and volume losses are equivalent to duodenal acid load ad emptying rate, respectively. The method has been validated by virtually abolishing the endogenous secretion of acid (by i

Error-Prone Replication of a 5-Formylcytosine-Mediated DNA-Peptide Cross-Link in Human Cells

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C--\u3eT transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol iota or hPol zeta reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol eta and hPol kappa with knockdown of hPol zeta, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol delta and hPol can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases