Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the HgII-mediated T-T (T-HgII-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of HgII. A characteristic Raman marker band at 1586 cm−1 was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift (18O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4−) is involved as a resonance contributor in the thymine ring of the T-HgII-T pair. This resonance includes the N-HgII-bonded state (HgII-N3-C4=O4) and the N-HgII-dissociated state (HgII+ N3=C4-O4−), and the latter contributor reduced the bond order of N-HgII. Consequently, the HgII nucleus in the T-HgII-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.

We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.

Sodium glycocholate, sodium taurocholate, sodium deoxycholate, EDTA, sodium salicylate, sodium caprate, diethyl maleate, N-lauryl-beta-D-maltopyranoside, linoleic acid polyoxyethylated (60 mol) mixed micelles (all 20 mM) have been ranked in order of their effectiveness as enhancers of the absorption of drugs in the rat small intestine, by use of an in-situ loop model with phenol red as a model drug. Local toxicity in rats was examined by assessing protein and phospholipid release as biological markers. Of the absorption enhancers, sodium deoxycholate, EDTA and N-lauryl-beta-D-maltopyranoside were the most effective; sodium deoxycholate and EDTA, however, caused significant release of protein and phospholipids. N-lauryl-beta-D-maltopyranoside, on the other hand, did not damage the small intestinal membrane. Sodium taurocholate enhanced phenol red absorption from the small intestine and resulted in little or no protein and phospholipids release. Sodium salicylate, diethyl maleate and the mixed micelles had no absorption-promoting effects on phenol red. There was good correlation between the area under the plasma concentration-time curve for phenol red and the amounts of protein and phospholipid released in the presence of absorption enhancers. From these results it might be concluded that N-lauryl-beta-D-maltopyranoside and sodium taurocholate are effective absorption enhancers which have low toxicity levels at a concentration of 20 mM.

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C-and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed ϳ40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A-and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.The human dipeptide transporter, hPepT1, is a 708-amino acid protein containing 12 ␣-helical transmembrane segments.