Removal of the impurities from environmental water samples for tritium measurement by liquid scintillation counting

Liquid scintillation counting is the most popular method for tritium measurement, however, it takes much time and a lot of doing to distill off the impurities before mixing the sample water and liquid scintillation cocktail. We have investigated the possibility of an alternative method to the distillation. We have found out that the filtration can be an alternative to distillation for the environmental water samples before electrolytic enrichment.     

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response–Inducing Efficacy of Sustained Antigen Release Alone

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy‐to‐use, non‐immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real‐life infections. Recombinant elastin‐like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross‐linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd‐containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next‐generation vaccines.     

Ab initio protein structure prediction with force field parameters derived from water-phase quantum chemical calculation

Molecular dynamics (MD) simulations are extensively used in the study of the structures and functions of proteins. Ab initio protein structure prediction is one of the most important subjects in computational biology, and many trials have been performed using MD simulation so far. Since the results of MD simulations largely depend on the force field, reliable force field parameters are indispensable for the success of MD simulation. In this work, we have modified atom charges in a standard force field on the basis of water-phase quantum chemical calculations. The modified force field turned out appropriate for ab initio protein structure prediction by the MD simulation with the generalized Born method. Detailed analysis was performed in terms of the conformational stability of amino acid residues, the stability of secondary structure of proteins, and the accuracy for prediction of protein tertiary structure, comparing the modified force field with a standard one. The energy balance between alpha-helix and beta-sheet structures was significantly improved by the modification of charge parameters.     

Total synthesis of mycestericin A

The first total synthesis of mycestericin A (1) starting from tartrates is described. The Overman rearrangement of an allylic trichloroacetimidate generated a tetra-substituted carbon with nitrogen, and subsequent stereoselective transformations afforded the highly functionalized vinyl iodide. The cross-coupling of the vinyl iodide with a chiral organozinc species under Negishi conditions, followed by deprotection, completed the total synthesis of 1.     

Amino acid residues in GRK1/GRK7 responsible for interaction with S-modulin/recoverin

GRK1 is a visual pigment kinase in rods and is essential for inactivation of light-activated rhodopsin. The GRK1 activity is inhibited by binding of the Ca(2+)-bound form of S-modulin/recoverin. We previously identified the S-modulin/recoverin site to interact with GRK1. In the present study, we identified its counterpart in GRK1. We synthesized 29 of GRK1 or GRK7 partial peptides that cover the entire sequence of GRK1/GRK7, and examined whether these peptides inhibit S-modulin/recoverin activity most probably by preoccupying the binding site for GRK1. The inhibition was the greatest with the N-terminal peptide (p1, aa 3-23 in GRK7). On mutation of each of eight amino acid residues highly conserved in the p1 region of more than 10 orthologs, the inhibition was significantly reduced in the mutation of Leu(6), Asn(12) and Tyr(15). We further examined the binding of the peptides, including mutated ones, to S-modulin/recoverin with a resonance mirror biosensor. The binding correlated well with the degree of the inhibition by a peptide. The inhibition, therefore, seemed to be due to a direct binding of the kinase peptide to the binding site of active S-modulin/recoverin. A GRK1 region close to its C-terminus also seemed to be the binding site for S-modulin/recoverin.     

Aldehyde and ketone syntheses using methylthiomethyl p-tolyl sulfone

Methylthiomethyl p-tolyl sulfone (1) was conveniently alkylated to give mono- and dialkylated products (2 and 4). Reaction conditions for the trasformation of 2 and 4 into aldehydes (3) and ketones (5), respectively, were exploited.     

Direct capillary gas chromatographic analysis and thermal stability of bile acid esters of glucose

Summary This paper deals for the first time with a direct method for analysis of the α and β anomers of bile acid esters of glucose by capillary gas chromatography (CGC) without the need for a hydrolytic step. The bile acid esters were derivatized to their trimethylsilyl (TMS) ethers, which in turn were chromatographed on a short (7m) metal capillary column chemically coated with a thin (0.15 μm) film of thermostable, non-polar polydimethylsiloxane. Satisfactory CGC separation of the isomeric bile acid esters was achieved on the column; the β anomers eluted before the corresponding α isomers. Particularly noteworthy is that the α anomers are partially isomerized to the corresponding β anomers, and that both anomers are partially decomposed during CGC analysis, demonstrating the chemical specificity and thermal instability of the bile acid esters.     

Copolymerization of Carbon Monoxide with Cyclic Compounds Catalyzed by Transition Metal Complexes

This article reviews recent discovery and progress of copolymerization of CO with cyclic monomers such as methylenecyclopropanes and propylene oxide promoted by transition metal complexes. 2-Aryl-1-methylenecyclopropanes react with CO in the presence of the Pd catalyst under mild conditions (1 atm, room temperature) to afford the polyketones. The reaction involves alternating insertion of the two monomers and opening of the three-membered ring of the cyclic monomers. Mechanistic study of the polymer growth revealed that 2-aryl-1-methylenecyclopropane undergoes 1,2-insertion into the Pd–acyl bond and subsequent -alkyl elimination of the cyclopropylmethylpalladium group. A bicyclic methylenecyclopropane, 7-methylenebicyclo[4.1.0]heptane, also reacts with CO in the presence of the Pd complexes with diimine ligands to afford the polyketone with six-membered rings in the repeating unit. The NMR spectra of the polyketone indicate the regulated tacticity of the monomer units. Co complexes and a mixture of Co and Ru complexes with carbonyl ligands promote the copolymerization of CO with propylene oxide in the presence of amine additives under CO pressure at 50 atm. Structures of the produced polyesters vary depending on the reaction conditions.     

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines.

The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50 mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of λ_ex = 415 nm and λ_em = 485 nm. A wide, linear relationship was obtained between the concentration of urea (1.0–100 μM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.     

Duplex dissociation of telomere DNAs induced by molecular crowding

Because of the importance of telomere DNAs, the structures of these DNAs in vivo are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. To understand the structure of biomolecules in vivo, their properties studied in vitro are extrapolated to the in vivo condition, while the condition in a living cell is inherently molecularly crowded and a nonideal solution contains various biomolecules. We investigated the effect of molecular crowding, which is one of the most important cellular environmental conditions, on the structure and stability of the telomere and G-rich and C-rich DNAs using circular dichroism (CD) spectra, CD melting curves, and isothermal titration calorimetry (ITC). The CD spectra and CD melting curves of G-rich DNA, C-rich DNA, and the 1:1 mixture of G-rich and C-rich DNAs showed that each G-rich DNA, C-rich DNA, and the 1:1 mixture form the antiparallel G-quadruplex, I-motif, and duplex, respectively, in the noncrowding condition as previously considered. On the contrary, the G-rich and C-rich DNAs individually form the parallel G-quadruplex and I-motif, respectively, in the molecular crowding condition, and the 1:1 mixture folds into the parallel G-quadruplex and I-motif but does not form a duplex. The ITC measurements indicated that the thermodynamic stability (DeltaG degrees (20)) of the duplex formation between the G-rich and C-rich DNAs in the noncrowding condition was -10.2 kcal mol(-)(1), while only a small heat change was observed in the ITC measurements in the molecular crowding condition. These ITC results also demonstrated that the molecular crowding condition prevents any duplex formation between G-rich and C-rich DNAs. These results indicate that a structural polymorphism of the telomere DNAs is induced by molecular crowding in vivo.