Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Preparation and characterization of stable dispersions of carbon black and nanodiamond in culture medium for in vitro toxicity assessment

Stable dispersions of carbon black and nanodiamond in culture medium were prepared by adding a pre-mixed dispersion of commercial carbon black or nanodiamond in aqueous bovine serum albumin (BSA) solution to culture medium. Dynamic light scattering revealed that carbon black and nanodiamond dispersions prepared in both NaCl solution and culture medium were highly stable. From DLVO theory and the results of zeta potential measurements, the theoretical effect of the electrostatic interactions between adsorbed BSA molecules was found to be minimal. The asymmetric flow field-flow fractionation measurements revealed that 0.05 or 0.56 mg/mL of BSA molecules were adsorbed on 0.11 or 1.09 mg/mL of carbon black, respectively, indicating 1:2 complexation of BSA with the colloidal particles. In the case of nanodiamond, 0.06 or 0.60 mg/mL of BSA molecules were adsorbed on 0.10 or 1.05 mg/mL of nanodiamond, respectively, indicating 1:2 complexation of BSA, which is the same ratio as in the case of carbon black. The adsorbed BSA molecules served as an effective stabilizing agent for the carbon black and nanodiamond, ensuring dispersion stability for at least 1 week. The preparation of the dispersions can be easily carried out by other researchers for toxicity studies.     

Pressure-drop reduction and heat-transfer deterioration of slush nitrogen in horizontal pipe flow

Cryogenic slush fluids such as slush hydrogen and slush nitrogen are two-phase, single-component fluids containing solid particles in a liquid. Since their density and refrigerant capacity are greater than those of liquid-state fluid alone, there are high expectations for the use of slush fluids in various applications such as clean-energy fuels, spacecraft fuels for improved efficiency in transportation and storage, and as refrigerants for high-temperature superconducting equipment. Experimental tests were performed using slush nitrogen to obtain the flow and heat-transfer characteristics in two different types of horizontal circular pipes with inner diameters of 10 and 15 mm. One of the primary objectives for the study was to investigate the effect of pipe diameter on the pressure-drop reduction and heat-transfer deterioration of slush nitrogen according to changes in the pipe flow velocity, solid fraction and heat flux. In the case of an inner diameter of 15 mm, pressure drop was reduced and heat-transfer characteristics deteriorated when the pipe flow velocity was higher than 3.6 m/s. On the other hand, in the case of an inner diameter of 10 mm, pressure drop was reduced and heat-transfer characteristics deteriorated when the pipe flow velocity was higher than 2.0 m/s. From these results, it can be seen that a larger pipe diameter produces a higher onset velocity for reducing pressure drop and deteriorating heat-transfer characteristics. Furthermore, based on observations using a high-speed video camera, it was confirmed that pressure drop was reduced and heat-transfer characteristics deteriorated when the solid particles migrated to the center of the pipe and the flow pattern of the solid particles inside the pipe was pseudo-homogeneous.     

Effects of catalyst and vehicle in coal liquefaction

Experiments have been carried out, using a semi-batch reactor equipped with a consecutive sampling device, to clarify the effects of catalysts and vehicles in the coal liquefaction process. The results show that a vehicle has a significant effect during preheating, unlike a catalyst which is not effective at this stage. A catalyst is more effective in promoting liquefaction under reaction conditions of 450°C and ≈ 20 MPa than is a vehicle. A vehicle higher in hydrogen donation increases the yield of oil even if a catalyst is present, providing a multiplier effect. In the development of a direct coal liquefaction process, therefore, selection of a vehicle is as important as that of a catalyst.     

Hydrodynamics characterization of the Maxblend impeller

The hydrodynamic characteristics of the Maxblend^TM impeller have been investigated in the case of viscous Newtonian fluids. Both laboratory experiments and 3D finite element based computational fluid dynamics (CFD) simulations have been carried out. The power consumption, the mixing evolution yielding the mixing time, and the effect of baffles in the laminar and transition flow regimes have been determined. It was found that the limit Reynolds number between the laminar and transition regimes is approximately 25 and 38 for the unbaffled and baffled configurations, respectively. Based on the range of Reynolds numbers studied in this work, the best window performance of the Maxblend^TM mixer where fast and homogenous mixing is achieved is the end of the laminar regime and the early transition regime with baffles.     

High drug loading self-microemulsifying/micelle formulation: design by high-throughput formulation screening system and in vivo evaluation

Purpose: To design a high drug loading formulation of self-microemulsifying/micelle system. Methods: A poorly-soluble model drug (CH5137291), 8 hydrophilic surfactants (HS), 10 lipophilic surfactants (LS), 5 oils, and PEG400 were used. A high loading formulation was designed by a following stepwise approach using a high-throughput formulation screening (HTFS) system: (1) an oil/solvent was selected by solubility of the drug; (2) a suitable HS for highly loading was selected by the screenings of emulsion/micelle size and phase stability in binary systems (HS, oil/solvent) with increasing loading levels; (3) a LS that formed a broad SMEDDS/micelle area on a phase diagram containing the HS and oil/solvent was selected by the same screenings; (4) an optimized formulation was selected by evaluating the loading capacity of the crystalline drug. Aqueous solubility behavior and oral absorption (Beagle dog) of the optimized formulation were compared with conventional formulations (jet-milled, PEG400). Results: As an optimized formulation, d-α-tocopheryl polyoxyethylene 1000 succinic ester: PEG400?=?8:2 was selected, and achieved the target loading level (200?mg/mL). The formulation formed fine emulsion/micelle (49.1?nm), and generated and maintained a supersaturated state at a higher level compared with the conventional formulations. In the oral absorption test, the area under the plasma concentration-time curve of the optimized formulation was 16.5-fold higher than that of the jet-milled formulation. Conclusions: The high loading formulation designed by the stepwise approach using the HTFS system improved the oral absorption of the poorly-soluble model drug.     

Isolation and quantitative detection of tetrachloroethene (PCE)-dechlorinating bacteria in unsaturated subsurface soils contaminated with chloroethenes

The estimation of tetrachloethene (PCE) dechlorinating-activity and identification of PCE-dechlorinating bacteria were performed in 65 unsaturated subsurface soils (at a depth 30-60 cm) that were collected from 21 noncontaminated soils and 44 chloroethene-contaminated soils including four soils that dechlorinated PCE to 1,2-cis-dichloroethene (cisDCE) in situ. Sixteen out of the 44 PCE-contaminated soils and three out of the 21 noncontaminated soils dechlorinated PCE to trichloroethene and cisDCE but not vinyl chloride or ethene after a month of incubation with 0.1% yeast extract at 30 degrees C. Desulfitobacterium sp. strain B31e3 that can dechlorinate PCE to cisDCE was isolated from a soil that dechlorinated PCE to cisDCE in situ. 16S rRNA gene of this strain showed the closest similarity of 99.1% with that of Desulfitobacterium hafniense (formally frappieri) strain DP7. Real-time PCR using specific primer sets targeted to the 16S rRNA genes of the representative PCE-dechlorinating bacteria, Dehalococcoides spp., Desulfitobacterium spp., and Dehalobacter spp. were performed using five unsaturated subsurface soils that dechlorinated PCE and three that did not dechlorinate PCE. In two out of the five soils that dechlorinated PCE, Desulfitobacterium spp. (0.12, 0.38% of total bacteria) and Dehalobacter spp. (0.0045, 0.0061% of total bacteria) were detected, and in one of the five soils, only Desulfitobacterium spp. (0.042% of total bacteria) was detected. None of these representative PCE-dechlorinating bacteria were detected in two out of the five soils that dechlorinated PCE and in all of the three soils that did not dechlorinate PCE. Dehalococcoides spp. were not detected in any unsaturated subsurface soils used in this study. These results suggested the involvement of Desulfitobacterium spp. and probably Dehalobacter spp. rather than Dehalococcoides spp. in the dechlorination of PCE to cisDCE in unsaturated subsurface soils.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.     

Residue analysis of metoclopramide in bovine and swine tissues by HPLC and administration experiment

A reversed-phase ion-pair HPLC method with ultraviolet detection has been developed for determination of metoclopramide (MCP) in bovine and swine muscle, liver, kidney, fat and intestine. MCP was extracted from samples with acetonitrile, and the extracts were cleaned up on an Oasis HLB cartridge (60 mg) after liquid-liquid extraction. The limit of detection of MCP was 0.002 microg/g and the limit of quantitation was 0.007 microg/g. Recoveries of MCP spiked at 0.03 ppm ranged from 74.1 to 93.3% for bovine tissues and from 86.1 to 92.7% for swine tissues. The present method was used for the analysis of bovine and swine tissues 1 day after withdrawal following drug administration. The MCP concentrations in all tissues were lower than the Japanese provisional MRLs.