Cation-pi interaction in the polyolefin cyclization cascade uncovered by incorporating unnatural amino acids into the catalytic sites of squalene cyclase

It has been assumed that the pi-electrons of aromatic residues in the catalytic sites of triterpene cyclases stabilize the cationic intermediates formed during the polycyclization cascade of squalene or oxidosqualene, but no definitive experimental evidence has been given. To validate this cation-pi interaction, natural and unnatural aromatic amino acids were site-specifically incorporated into squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius and the kinetic data of the mutants were compared with that of the wild-type SHC. The catalytic sites of Phe365 and Phe605 were substituted with O-methyltyrosine, tyrosine, and tryptophan, which have higher cation-pi binding energies than phenylalanine. These replacements actually increased the SHC activity at low temperature, but decreased the activity at high temperature, as compared with the wild-type SHC. This decreased activity is due to the disorganization of the protein architecture caused by the introduction of the amino acids more bulky than phenylalanine. Then, mono-, di-, and trifluorophenylalanines were incorporated at positions 365 and 605; these amino acids reduce cation-pi binding energies but have van der Waals radii similar to that of phenylalanine. The activities of the SHC variants with fluorophenylalanines were found to be inversely proportional to the number of the fluorine atoms on the aromatic ring and clearly correlated with the cation-pi binding energies of the ring moiety. No serious structural alteration was observed for these variants even at high temperature. These results unambiguously show that the pi-electron density of residues 365 and 605 has a crucial role for the efficient polycyclization reaction by SHC. This is the first report to demonstrate experimentally the involvement of cation-pi interaction in triterpene biosynthesis.     

Evaluation of the disintegration properties of commercial famotidine 20 mg orally disintegrating tablets using a simple new test and human sensory test

The purpose of this study was to demonstrate the usefulness and broad-applicability of a simple disintegration test method for orally disintegrating tablets (ODT). Eight types of commercial famotidine 20 mg orally disintegrating tablets with different physical properties (formulation, manufacturing method, tablet weight, shape, diameter, thickness, etc.), were used. Disintegration times of these tablets were evaluated employing human sensory test, conventional disintegration test, and the new proposed disintegration test. The human sensory test was performed in 5 healthy volunteers. In the conventional disintegration test, the disintegration apparatus described in the Japanese Pharmacopeia (JP 1(st)) was used. Our proposed new test which is characterized by a rotating shaft with a low weight (10, 15 g) and rotation speed (10, 25, 50 rpm) was evaluated using tablets with and without storage under severe conditions (60 degrees C/75%RH for 1 week). The disintegration times of famotidine 20 mg orally disintegrating tablets in human sensory test varied from 9 to 32 s. In contrast, disintegration times in the conventional test were prolonged to over 300 s. Disintegration times in the new proposed test were close to those in human sensory test. Especially, when the new test was conducted with 15 or 10 g weight and 25 rpm, the slope (human sensory test vs. new proposed test) was almost 1. We were able to demonstrate that the new proposed test was useful to estimate the actual human disintegration time.     

Syntheses and characterization of oxygen/sulfur-bridged incomplete cubane-type clusters, [Mo3S4Tp3]+ and [Mo3OS3Tp3]+, and a mixed-metal cubane-type cluster, [Mo3FeS4ClTp3]. X-ray structures of [Mo3S4Tp3]Cl, [Mo3OS3Tp3]PF6, and [Mo3FeS4ClTp3

The reaction of [Mo3S4(H2O)9]4+ (1) with hydrotris(pyrazolyl)borate (Tp) ligands produced [Mo3S4Tp3]Cl x 4 H2O ([3]Cl x 4 H2O) in an excellent yield. An X-ray structure analysis of [3]Cl x 4 H2O revealed that each molybdenum atom bonded to the Tp ligand. We report four salts of 3, [3]Cl x 4 H2O, [3]tof x 2 H2O, [3]PF6 x H2O, and [3]BF4 x 2 H2O in this paper. The solubility and stability of the chloride salt in organic solvents differ completely from those of the other salts. We have also prepared a new compound, [Mo3OS3Tp3]PF6 x H2O ([4]PF6 x H2O), via the reaction of [Mo3OS3(H2O)9]4+ (2) with KTp in the presence of NH4PF6. All the molybdenum atoms bonded to Tp ligand. 1H NMR signals corresponding to nine protons bonded to three pyrazole rings in one Tp were observed in a spectrum (at 253 K) of [3]BF4 x 2 H2O. It shows that cluster 3 has a 3-fold rotation axis in CD2Cl2 solution. Twenty-one 1H NMR signals corresponding to twenty-seven protons bonded to nine pyrazole rings in three Tp ligands were observed in a spectrum (at 233 K) of [4]PF6 x H2O; obviously, 4 has no 3-fold rotation axis, in contrast to 3. The short CH...mu3S distance caused large upfield chemical shifts in the 1H NMR spectra of 3 and 4. The reaction of 3 with metallic iron in CH2Cl2 produced [Mo3FeS4XTp3] (X = Cl (5), Br (6)). X-ray structure analysis of 5 has revealed the existence of a cubane-type core Mo3FeS4. Complex 3 functions as a metal-complex ligand for preparing a novel mixed-metal complex even in nonaqueous solvents. The cyclic voltammogram of 5 shows two reversible one-electron couples (E(1/2) = -1.40 and 0.52 V vs SCE) and two irreversible one-electron oxidation processes (E(pc) = 1.54 and 1.66 V vs SCE).     

Guanidinium ylide mediated aziridination: identification of a spiro imidazolidine-oxazolidine intermediate

We successfully isolated a spiro imidazolidine-oxazolidine intermediate in the reaction of guanidinium ylide mediated aziridination using alpha-bromocinnamaldehyde. X-ray crystallographic analysis unambiguously revealed that the stereogenic centers of the spiro intermediate were in a trans configuration. The role of the spiro compound as an intermediate in the aziridination reaction was confirmed by observation of its smooth chemical conversion into aziridine products.     

A novel method for the separation of bis(alpha-hydroxyalkyl)phosphinic acid diastereoisomers via formation of novel cyclic phosphinic acids

The reaction of aromatic and aliphatic aldehydes with hypophosphorus acid under microwave irradiation was examined. The reaction gave a mixture of a racemic pair of bis(alpha-hydroxyalkyl)phosphinic acids and acetal derivatives from the corresponding bis(alpha-hydroxyalkyl)phosphinic acids of meso-stereochemistry in good yield. The difference in solubility in organic solvents due to polarity allowed us to readily separate these compounds. This method constitutes an easy, rapid, and good-yielding preparation and separation of bis(alpha-hydroxyalkyl) phosphinic acid diastereoisomers from simple starting materials using microwave irradiation.     

Simultaneous observation of CO isotopomer absorption by broadband difference-frequency generation using a direct-bonded quasi-phase-matched LiNbO3 waveguide

Rovibration absorption lines both of 12CO and 13CO are observed simultaneously with the output of a 2 microm broadband difference frequency generated in a direct-bonded quasi-phase-matched LiNbO3 waveguide, which is a 50 mm device with a single quasi-phase-matching period that is operated at a constant temperature. The wavelength conversion efficiency and the difference-frequency generation bandwidth reach 100%/W and 100 nm, respectively. The idler output bandwidth in the 2 microm region is obtained by group-velocity matching or phase-mismatch minimization when a 0.94 microm pump laser diode and a 1.55 microm tunable signal source are used.     

Analysis of a collinear holographic storage system: introduction of pixel spread function

Image formation in a collinear holographic storage system was analyzed. The wavefront from each pixel of a spatial light modulator was regarded as a plane wave in the recording medium, and its wave vector was determined by the position of the pixel. The hologram in the recording medium was treated as the summation of all gratings written by all combinations of two plane waves. The image of a data page was formed by diffraction of the reference waves by all gratings. The results of the simulation showed good agreement with experiment. We introduced the pixel spread function to describe the image formation characteristics. Analysis of the pixel spread function reveals that a radial-line pixel pattern for reference waves gave a sharper image than other reference pixel patterns. It is also shown that a random phase modulation applied to each reference pixel improved the image formation.     

Modified Vonk's Method to Determine Crystallinity and Crystal Distortion in Polymers

Using a modified method developed from Vonk's method, detailed values of crystallinity and crystal disorder were obtained by wide angle X-ray diffraction (WAXD). In Vonk's method, the crystallinity (w) is determined by extrapolation of the WAXD experimental curve back to zero scattering angle, while the distortion factor (k) is determined by the inclination of the experimental curve at zero scattering angle. In our new method, both w and k are determined at the same time by using the least squares method. In order to show the efficiency of our method, the new fitting procedure was applied to the experimental values of polyethylene and polyethylene terephthalate as measured by Vonk, and the values of w and k determined by our new method were compared with those found by Vonk's method. The new fitting method has the advantage that it uses the whole experimental curve. Moreover, our modified Vonk's method enables us to discuss crystal distortions as arising from first-kind (thermal) disorder and second-kind (paracrystalline) disorder.     

Microwave-assisted digestion with a single reaction chamber for mineral fertilizer analysis by inductively coupled plasma optical emission spectrometry

A systematic approach is proposed to evaluate microwave-assisted digestion in a single reaction chamber for the simultaneous determination of elements in the mineral fertilizer and raw material by inductively coupled plasma optical emission spectrometry. A reference material was digested using 12 acid mixtures containing nitric, hydrochloric, orthophosphoric, hydrofluoric or boric acid. Principal components analysis was applied for data treatment. Three digestion procedures based on diluted acids exhibited the most accurate results, with relative errors for reference values ranging from ?15% to +?9%. Limits of quantification were in the range of 0.2?mg cadmium kg^?1 up to 1000?mg aluminum kg^?1.     

Changes in diffusion tensor imaging (DTI) eigenvalues of skeletal muscle due to hybrid exercise training

Several studies have proposed the cell membrane as the main water diffusion restricting factor in the skeletal muscle cell. We sought to establish whether a particular form of exercise training (which is likely to affect only intracellular components) could affect water diffusion. The purpose of this study is to characterise prospectively the changes in diffusion tensor imaging (DTI) eigenvalues of thigh muscle resulting from hybrid training (HYBT) in patients with non-alcoholic fatty liver disease (NAFLD). Twenty-one NAFLD patients underwent HYBT for 30 minutes per day, twice a week for 6 months. Patients were scanned using DTI of the thigh pre- and post-HYBT. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), the three eigenvalues lambda 1 (λ1), λ2, λ3, and the maximal cross sectional area (CSA) were measured in bilateral thigh muscles: knee flexors (biceps femoris (BF), semitendinosus (ST), semimembranous (SM)) and knee extensors (medial vastus (MV), intermediate vastus (IV), lateral vastus (LV), and rectus femoris (RF)), and compared pre- and post-HYBT by paired t-test. Muscle strength of extensors (P < 0.01), but not flexors, increased significantly post-HYBT. For FA, ADC and eigenvalues, the overall picture was of increase. Some (P < 0.05 in λ2 and P < 0.01 in λ1) eigenvalues of flexors and all (λ1-λ3) eigenvalues of extensors increased significantly (P < 0.01) post-HYBT. HYBT increased all 3 eigenvalues. We suggest this might be caused by enlargement of muscle intracellular space.