Hydrogen Peroxide-Triggered Conversion of Boronic Acid-Appended Insulin into Insulin and Its Application as a Glucose-Responsive Insulin Formulation.

p-Boronophenylmethoxycarbonyl (BPmoc) is a protecting group for amines that is removable by treatment with hydrogen peroxide (H2O2). We prepared BPmoc-modified insulin (BPmoc-Ins) and subcutaneously injected the formulation into diabetic rats. The results demonstrated that BPmoc effectively sealed the blood glucose (Glc)-lowering effects of Ins. Conversely, coinjection of BPmoc-Ins and Glc oxidase (GOx) resulted in reduced blood Glc levels, indicating that Ins was generated from BPmoc-Ins through the following reactions: oxidation of endogenous Glc by GOx; production of H2O2 accompanied by Glc oxidation; removal of BPmoc residues by H2O2. These results show the potential of BPmoc-Ins for a Glc-responsive Ins release system.

Inhibitory effects of organophosphate esters on carboxylesterase activity of rat liver microsomes.

We investigated the inhibitory effects of 13 organophosphate esters (OPEs) and hydrolytic metabolites on the carboxylesterase activity of rat liver microsomes in vitro in order to examine whether there might be a potential impact on human health, and to elucidate the structure activity relationship. Among the test compounds, 2-ethylhexyl diphenyl phosphate (EDPhP) was the most potent inhibitor of carboxylesterase activity, as measured in terms of 4-nitrophenol acetate hydrolase activity, followed by tri-m-cresyl phosphate (TmCP), cresyl diphenyl phosphate (CDPhP) and triphenyl phosphate (TPhP). The IC50 values were as follows: EDPhP (IC50: 0.03 µM) > TmCP (0.4 µM) > CDPhP (0.8 µM) > TPhP (14 µM) > tris(1,3-dichloro-2-propyl) phosphate (17 µM) > tris(2-ethylhexyl) phosphate (77 µM) > tri-n-propyl phosphate (84 µM) > tris(2-chloroethyl) phosphate (104 µM) > tris(2-butoxyethyl) phosphate (124 µM) > tri-n-butyl phosphate (230 µM). The IC50 value of EDPhP was three orders of magnitude lower than that of bis(4-nitrophenyl) phosphate, which is widely used as an inhibitor of carboxylesterase. Trimethyl phosphate, triethyl phosphate and tris(2-chloroisopropyl) phosphate slightly inhibited the carboxylesterase activity; their IC50 values were above 300 µM. Lineweaver-Burk plots indicated that the inhibition by several OPEs was non-competitive. Diphenyl and monophenyl phosphates, which are metabolites of TPhP, showed weaker inhibitory effects than that of TPhP.

Biochemical, spectroscopic and X-ray structural analysis of deuterated multicopper oxidase CueO prepared from a new expression construct for neutron crystallography.

Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, from Escherichia coli is involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space group P21, with unit-cell parameters a = 49.51, b = 88.79, c = 53.95 Å, ß = 94.24°, and deuterated CueO crystallized in space group P212121, with unit-cell parameters a = 49.91, b = 106.92, c = 262.89 Å. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV-Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms in the active site to those of previously reported CueO structures. These results indicate that the CueO protein prepared using the new construct is suitable for further neutron diffraction studies.

Kefiran reduces atherosclerosis in rabbits fed a high cholesterol diet.

AIM: Kefiran is an exopolysaccharide produced by Lactobacillus kefiranofaciens, and has been proposed to have many health-promoting properties. We investigated the antiatherogenic effect of kefiran on rabbits fed a high-cholesterol diet. METHODS: Male New Zealand White rabbits were fed a 0.5% cholesterol diet without (control group, n = 7) or with kefiran (kefiran group, n = 8) for eight weeks. The aorta was analyzed by histochemistry and atherosclerotic lesions were quantified. Lipids and sugars in serum were measured. Foam cell formation of RAW264.7 by ßVLDL derived from both groups of rabbits was also investigated. RESULTS: Cholesterol, triglyceride and phospholipids levels of serum and lipoprotein fractions were not significantly different between these groups. Atherosclerotic lesions of the aorta in the kefiran group were statistically lower than those of the control group, with marked differences in the abdominal aorta. T-lymphocytes were not detectable in the aorta of the kefiran group. Cholesterol contents in stools were almost identical in both groups. Cholesterol content in the liver of the kefiran group was statistically lower than in the control group. Galactose content of ßVLDL derived from the kefiran group was higher, and the lipid peroxidation level was much lower than in the control group. RAW264.7 macrophages treated with ßVLDL from the kefiran group showed a more spherical shape and accumulated statistically lower cholesterol than macrophages treated with ßVLDL from the control group. CONCLUSION: Orally derived kefiran is absorbed in the blood. Kefiran prevents the onset and development of atherosclerosis in hypercholesterolemic rabbits by anti-inflammatory and anti-oxidant actions.

Chiral recognition in cucurbituril cavities.

For the first time, achiral cucurbiturils (CBs) were endowed with significant enantiomeric and distereomeric discrimination by incorporating a strong chiral binder. Calorimetric, nuclear magnetic, light-scattering, and mass spectral studies revealed that (S)-2-methylbutylamine (as a strong binder) can be discriminated by two enantiomeric supramolecular hosts, composed of CB[6] and (R)- or (S)-2-methylpiperazine, with an unprecedented 95% enantioselectivity in aqueous NaCl solution. This is the highest enantioselectivity ever reported for a supramolecular system derived from an achiral host. Similarly, CB[7], with a larger cavity, exhibited diastereoselectivities up to 8 times higher for diastereomeric dipeptides, as demonstrated for L-Phe-L-Leu-NH3+ versus L-Phe-D-Leu-NH3+.