Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Evaluation of the disintegration time of rapidly disintegrating tablets via a novel method utilizing a CCD camera

Many kinds of rapidly disintegrating or oral disintegrating tablets (RDT) have been developed to improve the ease of tablet administration, especially for elderly and pediatric patients. In these cases, knowledge regarding disintegration behavior appears important with respect to the development of such a novel tablet. Ordinary disintegration testing, such as the Japanese Pharmacopoeia (JP) method, faces limitations with respect to the evaluation of rapid disintegration due to strong agitation. Therefore, we have developed a novel apparatus and method to determine the dissolution of the RDT. The novel device consists of a disintegrating bath and CCD camera interfaced with a personal computer equipped with motion capture and image analysis software. A newly developed RDT containing various types of binder was evaluated with this protocol. In this method, disintegration occurs in a mildly agitated medium, which allows differentiation of minor distinctions among RDTs of different formulations. Simultaneously, we were also able to detect qualitative information, i.e., morphological changes in the tablet during disintegration. This method is useful for the evaluation of the disintegration of RDT during pharmaceutical development, and also for quality control during production.     

Novel approach to arylhydrazones, the precursor for Fischer indole synthesis, via diazo esters derived from α-amino acid esters

A novel method for synthesizing arylhydrazones, the precursor for Fischer indole synthesis, using aryllithium reagents and α-diazo esters that are easily obtained from α-amino acid esters, is described.     

Structural change of 1-butyl-3-methylimidazolium tetrafluoroborate + water mixtures studied by infrared vibrational spectroscopy

Mixtures of ionic liquid (IL, 1-butyl-3-methylimidazolium tetrafluoroborate, [BMIM][BF4]) and water with varying concentrations were studied by attenuated total reflection infrared absorption and Raman spectroscopy. Changes in the peak intensities and peak positions of CHx (x = 1, 2, 3) vibration modes of the cation of the IL and OH vibration modes of the water molecules were investigated. Peaks from normal-mode stretch vibrations of CH bonds belonging to the imidazolium ring of the cation did not change their positions, while those from the terminal methyl group of the butyl chain blueshifted by approximately 10 cm-1 with the addition of water. On the other hand, change in the spectral shape in the OH stretch vibration region shows hydrogen-bonding network of water molecules breaking down rapidly as the IL is added. Trends in the change of the peak positions and the peak intensities suggested qualitative change of the intermolecular structure in the [BMIM][BF4] + H2O mixture at 32 +/- 2 and 45 +/- 2 mol/L of water concentration.     

Analyses of the EPR responses of sucrose and L-alpha-alanine radicals induced by C, Ne, and Ar ion irradiations

Using electron paramagnetic resonance (EPR), we investigated the signal responses of sucrose and L-alpha-alanine radicals produced by heavy-ion irradiation with various linear energy transfers (LETs) and absorbed dose. The spin yields for the two compounds showed a linear relation with the absorbed dose, as well as a logarithmic correlation with the LET. A quantitative EPR analysis showed that sucrose was more sensitive to the particle species than that of alanine. Hence, the present EPR results imply that sucrose can be useful as a radiation indicator. Further analysis was carried out for the radical-production cross section, which showed that stable radicals of the two compounds were produced through collisions of several particles with a single molecule.     

Simultaneous Determination of Imidazole Amino Acids, Aromatic Amino Acids, and Creatinine by Dual-Mode Gradient HPLC Using a Low-Capacity Sulfo-Functionalized Poly(Ethylstyrene-Divinylbenzene) Resin Column

This paper presents a low-capacity cation-exchange chromatography method for the analysis of UV-absorbing dipeptides and amino acids. A newly marketed low-capacity cation-exchange column packed with sulfo-functionalized highly cross-linked macroreticular poly(ethylstyrene-divinylbenzene) copolymer was used for the simultaneous determination of imidazole amino acids, aromatic amino acids, and creatinine in urine samples. A dual-mode binary gradient chromatography method was established using two solvents, A: 15 mM H₃PO₄/5 mM ethylenediamine and B: 15 mM H₃PO₄/5 mM ethylenediamine/40 (v/v) % CH₃CN at 40 °C, with an optimized time program for changing the delivery ratio of A/B and the flow rate. Good chromatograms were obtained within an acceptable cycle time of 25 min. The quantification data were satisfactory for all analytes, showing the relative standard deviations (RSD) of retention times between 0.08 and 1.68 %; RSDs of area intensities between 0.23 and 2.60 %; and linear regression lines with r ² more than 0.9994. The method could determine the creatinine ratios of the diagnostic markers on the single chromatographic run, which enabled to discriminate disease from health. For example, the creatinine ratios for phenylketonuria were significantly higher than those for controls. The method can provide highly cost-efficient information or useful knowledge for clinical and pharmaceutical studies.