Very high solubility and low enthalpy of dissolution of orthorhombic lysozyme crystals grown in deionized water

Solubility data are indispensable to control the quality of protein crystals. The solubility curves of orthorhombic hen egg-white lysozyme (HEWL) crystals grown in deionized water (salt-free crystals) were measured in this study. Once salt-free crystals have been successfully grown, we can measure solubility with arbitrary salt concentrations, including in vivo. The absolute values of the solubilities of the salt-free crystals were found to be the largest of all HEWL crystals grown under various high salt concentration conditions. Van t Hoff plots of the solubilities of the salt-free crystals showed the lowest enthalpy of dissolution (15 ± 2 kJ mol-1).


In-situ observation of homogeneous nucleation of colloidal crystals formed with attractive interactions under density-matching conditions

Colloidal crystals formed with attractive interactions are used for in-situ observations ofhomogeneous nucleation processes in density-matched dispersion.We found globular and plate-likepolycrystalline clusters stably suspended in the dispersion. Both clusters rotated randomlyand moved freely with suspending in the dispersion. They were probably formed through homogeneous nucleation processes.


Solution-mediated phase transition of protein crystals

A snapshot of the evidence of solution-mediated phase transition of glucose isomerase (GI)crystals more than 7 months after the start of an experiment was successfully captured. The transition to P21212 crystals started about 2 months after the first I222 crystals nucleated. To conduct such long-term experiments, we developed novel observation cells with liquid paraffin layers to prevent aqueous GI solutions from evaporation of water. Changes in the weights of cells were measured, and much less evaporation was successfully achieved by continuously adding liquid paraffin as a liquid sealant.


Activation energy of kink incorporation of particles into colloidal crystals with attractive interactions

The kink incorporation and diffusion of particles on the growth interface of colloidal crystals with attractive interactions between particles are important elementary processes. We have successfully estimated the activation energy of kink incorporation, εkink, and vibrational frequency, ν, of silica particles on the growth interface using two fundamentally different approaches. One was the use of the Arrhenius plot of step velocities, Vstep, at five temperatures. The other was the use of ν measured by the direct observation of particle movements with a high-speed camera. Although ν and εkink took similar values for both approaches, the errors of ν and εkink are smaller for the direct observation case than those obtained by the Arrhenius plot. This study is the first to estimate quantitively the activation energy of kink incorporation and the vibrational frequency of particles on the growth interface of a colloidal crystal. These findings would contribute significantly to the understanding of crystal growth mechanisms.


Highly Purified Glucose Isomerase Crystals Under Microgravity Conditions Grow as Fast as Those on the Ground Do

Suppression of convection flows (solute transportation) and that of impurity incorporation into crystals seem to be the main reasons why the quality of protein crystals becomes better under microgravity conditions, whereas each precise mechanism has not been completely clarified yet. We tried to clarify the former reason by the in-situ observation of spiral growth hillocks on the {110} faces of highly purified glucose isomerase (GI) crystals under microgravity conditions and on the ground. Lateral growth rates Vlateral of a spiral hillock on the {110} face of a glucose isomerase crystal in situ under microgravity conditions and step velocities Vstep of the same configuration on the ground took similar values as far as the maximum values are compared each other. This similarity indicates there are less influences of the convection flows on the growth rates of protein crystals contrary to conventional expectations.


Precipitant-free Crystallization of Lysozyme and Glucose Isomerase by Drying

Protein crystallization is usually conducted by using precipitants, although the salting-out phenomenon is still unclear and complex. Moreover, the addition of precipitants sometimes results in irreversible disordered precipitation of protein molecules. Although precipitant-free lysozyme crystals obtained by centrifugal concentration showed significant changes in three-dimensional structure compared to the structure of salted-out crystals, it was rather difficult to mount crystals from a viscous dense liquid phase after centrifugal concentration, and the quality of the crystals often deteriorated during the mounting process. Here we present novel precipitant-free crystallization methods, which were effective for lysozyme and glucose isomerase. Tetragonal lysozyme crystals were successfully crystallized in a glass capillary simply by drying highly concentrated lysozyme solution in the presence of 0.01 M hydrochloric acid without using any precipitants. Glucose isomerase dissolved in ultra-pure water was also successfully crystallized in hanging drops by drying highly concentrated solution under low-humidity conditions. Oscillation images of the obtained crystals were safely collected without handling; they clearly indicated the crystals had a tetragonal form for lysozyme and an orthorhombic form for glucose isomerase, and their lattice parameters are similar to those of previously reported crystals obtained by salting-out methods.


Control of strain in subgrains of protein crystals by the introduction of grown-in dislocations

It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structure of protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals even though the overall crystal quality is rather high, as shown by clear equal thickness fringes in X-ray topography. Thus, a large stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals to radiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in the crystal. This suggests that the introduction of dislocations in protein crystals can be effective inenhancing the crystal quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased, leading to the collection of X-ray diffraction data sets with high diffractivity.

Acta Crystallographica. Section D, Structural Biology


タンパク質結晶成長場及びX線回折実験用試料固定材としてのトラックエッチド メンブランフィルターの利用


A novel handling-free method of mounting single protein crystals for synchrotron structure analyses at room temperature

We have developed a handling-free mounting method for X-ray crystallography ofprotein crystals at room temperaturesthe glass capillary method. In this method,crystals were nucleated and grown on the capillary walls, and then growth solutions weregently removed. The procedures for collecting data on the crystals were conducted bysimply setting the capillary on the goniometer of a synchrotron beamline withouttouching the crystals. Crystal quality was characterized using mosaicity, resolution at I /σ(I) = 2, I / σ(I) at resolution = 2.0 , Rmerge , and Completeness. Wilson plots were alsoused to characterize the quality of crystals. In particular, all samples showed very lowmosaicity; the handling-free method successfully retained their low mosaicity andeffectively maintained the crystal quality.


Effect of density change at crystallization on a one-dimensional heat balance equation at solid-liquid interface

Japanese Journal of Applied Physics, Part 1 (Regular Papers & Short Notes)


Very low nucleation rates of glucose isomerase crystals under microgravity in the International Space Station

In situ observation of the nucleation and growth of glucose isomerase (GI) crystals under microgravity was conducted using an optical microscope during the first flight of the Advanced Nano Step project undertaken in the International Space Station (ISS). Very low apparent nucleation rates (J) of GI crystals in the solution and on the substrate of the growth container were confirmed compared with those on the ground. In particular, J of GI crystals in the solution were a few times lower than that on the substrate. The growth rates (R) of the {101} faces of GI crystals in the solution and on the substrate were measured. The very low nucleation rates allowed us to successfully measure R at a very high supersaturation region (up to ln(C/Ce)=6), at which R cannot be measured on the ground.


Dependence of Convection Flow Rates on Gravity around Growing Hen Egg-White Lysozyme Crystals

Convection flows induced by formation of solute depletion zone around growing protein crystals are visualized to confirm whether growth of crystals at a ceiling position (top wall of a growth container) can really suppress this convection flow effectively or not. First, we observed the convection flows around the crystal at the ceiling position at 1.0 G. Second, parabolic flight experiments revealed that flow rates of polystyrene particles (as marker particles, 500 nm in diameter) around a crystal at the ceiling position moved and thus do not indicate zero at high-gravity conditions, and the particles almost stop at zero gravity. Thus, stable microgravity experiments are still indispensable to attain complete convection-free conditions.

International Journal of Microgravity Science and Application


Petit-High Pressure Carbon Dioxide stress increases synthesis of S-Adenosylmethionine and phosphatidylcholine in yeast Saccharomyces cerevisiae

Petit-High Pressure Carbon Dioxide (p-HPCD) is a promising nonthermal technology for foods pasteurization.Cluster analysis of gene expression profiles of Saccharomyces cerevisiae exposed to various stresses exhibitedthat gene expression profile for p-HPCD stress (0.5 MPa, 25 °C) was grouped into a cluster including profilesfor Sodium Dodecyl Sulfate and Roundup herbicide. Both are detergents that can disorder membrane structurallyand functionally, which suggests that cell membrane may be a target of p-HPCD stress to cause cell growth inhibition. Through metabolomic analysis, amount of S-Adenosylmethionine (AdoMet) that is used as methyl donorto participate in phosphatidylcholine synthesis via phosphatidylethanolamine (PE) methylation pathway, wasincreased after p-HPCD treatment for 2 h. The key gene OPI3 encoding phospholipid methyltransferase that catalyzes the last two steps in PE methylation pathway was confirmed significantly induced by RT-PCR. Transcriptional expression of genes (MET13, MET16, MET10, MET17, MET6 and SAM2) related to AdoMet biosynthesis wasalso significantly induced. Choline as the PC precursor and ethanolamine as PE precursor in Kennedy pathwaywere also found increased under p-HPCD condition.We also found that amounts of most of amino acids involvingprotein synthesis were found decreased after p-HPCD treatment for 2 h. Moreover, morphological changes on cellsurface were observed by scanning electron microscope. In conclusion, the effects of p-HPCD stress on cellmembrane appear to be a very likely cause of yeast growth inhibition and the enhancement of PC synthesis couldcontribute to maintain optimum structure and functions of cell membrane and improve cell resistance toinactivation.


Characterization of grown-in dislocations in high-quality glucose isomerase crystals by synchrotron monochromatic-beam X-ray topography

High quality glucose isomerase (GI) single crystals are grown by using chemical cross-linked seed crystals. The crystal structure is an orthorhombic system in which the molecular arrangement is close to a body-centered cubic (bcc) one. The crystal defects, especially dislocations, in GI crystals are experimentally characterized by synchrotron monochromatic-beam X-ray topography. Two straight dislocations are clearly observed, which originate from the interface between the cross-linked seed crystal and the grown crystal. From the invisibility criterion of the dislocation images, it is experimentally identified that they are close to be of pure edge character with the Burgers vector of [1 1 1] which is typical one in bcc metal crystals. Moreover, bead-like contrasts along the dislocation images and the equal-thickness fringes, related to Pendellösung fringes, at crystal edges are clearly observed, which have never been observed in other protein crystals so far. These contrasts can attributed to the dynamical diffraction effect which has been often observed in high-quality crystals such as Si. Thus it seems that the perfection of GI crystals shown in this paper is extremely high compared with other protein crystals reported so far.


Urea cycle is enhanced by petit-high pressure carbon dioxide stress in yeast Saccharomyces cerevisiae

It has been demonstrated that pasteurized effect on microorganisms of petit-high pressure carbon dioxide (p-HPCD) with long time treatment is similar to the effect of HPCD with short time treatment. The petit-high pressure refers to a pressure of 1.513atm (standard atmosphere). After 0.5MPa of CO2 at 25°C for 2h treatment, specific growth rate of yeast cells in the logarithm phase was decreased by 50% approximately. Under this condition, our study analyzed transcriptional responses of Saccharomyces cerevisiae through the functional genomic approach. Transcription of 837 open reading frames (ORFs) was altered relative to cells without treatment and 476 ORFs were induced after p-HPCD treatment. These selected genes were then categorized by function of gene product using the Munich Information Centre for Protein Sequences database. Genes involved in metabolism of the urea cycle were found to be significantly induced. This enhanced metabolic process could help to remove redundant HCO3 in cellular interior, thereby decrease the production of CO23. Liberation of H+ ion could be decreased along with the inhibition of HCO3 dissociation.


Importance of Determination of Crystal Quality in Protein Crystals when Performing High-Resolution Structural Analysis

We show that the extinction effect explained by the dynamical theory of diffraction must be considered even in the case of protein crystals. Equal-thickness fringes, which are attributed to the Pendello sung effect, were clearly observed in the region of a tapered glucose isomerase crystal with wedge-like edges using X-ray topography carried out with a beam of monochromatic synchrotron radiation. This indicates that the perfection of this glucose isomerase crystal is high enough to produce this dynamical theory-related effect: this surely leads to difficulty in the collection of accurate integrated intensities of diffraction spots for Xray structural analysis. Therefore, it is important to determine whether the crystal quality of a protein crystal under analysis is adequate to obtain accurate three-dimensional structures of protein molecules for X-ray structural analyses. We show that X-ray diffraction rocking-curve measurements can provide clues for this determination.


Adsorption, Desorption, Surface Diffusion, Lattice Defect Formation, and Kink Incorporation Processes of Particles on Growth Interfaces of Colloidal Crystals with Attractive Interactions

Good model systems are required in order to understand crystal growth processes because, in many cases, precise incorporation processes of atoms or molecules cannot be visualized easily at the atomic or molecular level. Using a transmission-type optical microscope, we have successfully observed in situ adsorption, desorption, surface diffusion, lattice defect formation, and kink incorporation of particles on growth interfaces of colloidal crystals of polystyrene particles in aqueous sodium polyacrylate solutions. Precise surface transportation and kink incorporation processes of the particles into the colloidal crystals with attractive interactions were observed in situ at the particle level. In particular, contrary to the conventional expectations, the diffusion of particles along steps around a two-dimensional island of the growth interface was not the main route for kink incorporation. This is probably due to the number of bonds between adsorbed particles and particles in a crystal; the number exceeds the limit at which a particle easily exchanges its position to the adjacent one along the step. We also found novel desorption processes of particles from steps to terraces, attributing them to the assistance of attractive forces from additionally adsorbing particles to the particles on the steps.


First Direct Observation of Impurity Effects on the Growth Rate of Tetragonal Lysozyme Crystals under Microgravity as Measured by Interferometry

The suppression of impurity effects on protein crystallization under microgravity would be related to the origin of the higher X-ray resolution limits observed for space-grown crystals, and the mechanism that the impurity effects are suppressed would be useful for producing high-quality protein crystals on the ground. The normal growth rates R and apparent step velocities (lateral growth rates of a spiral hillock) V of tetragonal hen egg-white lysozyme (HEWL) crystals were for the first time measured by Michelson interferometry in the international space station (as part of the NanoStep project) using commercialized HEWL samples containing 1.5% impurities. A significant increase in V under microgravity was confirmed compared to step velocities Vstep on ground, while a decrease in R was also confirmed compared to that in the purified solution under microgravity; therefore, even under microgravity, the purity level influences crystal growth processes as expected. More precisely, because of exact measurement of growth rates, kinetic analyses of R were conducted as a function of supersaturation, σ (σ ≡ ln(C/Ce), where C is the concentration of lysozyme, and Ce is the solubility), using a spiral growth model and a two-dimensional (2D) nucleation growth model. For both models over a wide range of σ, R in the impure solution was significantly lower than that in the purified solution. Thus, the final quality of the crystals would depend on the purity of the solutions even in microgravity environments. The degree of the suppression of impurity effects was also evaluated using the difference between apparent step velocities Vp and Vi, where Vp and Vi are the apparent step velocity in the purified and impure solution, respectively. The difference between Vp and Vi became significantly smaller than the difference in step velocities on the ground, Vstep,p and Vstep,i, where Vstep,p and Vstep,i are the step velocities in the pure and impure solution, respectively. Thus, the suppression of impurity effects under microgravity was confirmed for the first time.


Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station

The normal growth rates of the {110} faces of tetragonal hen egg white lysozyme (HEWL) crystals, R, was measured as a function of the supersaturation σ parameter using a reflection type interferometer under μG at the International Space Station (ISS, NanoStep Project). Since water slightly evaporated from in situ observation cells during a long term space station experiment for several months, equilibrium temperature Te changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration Cs. To correct σ, the actual Cs and protein concentration Cp, which correctly represent the measured Te value in space, were first calculated. Second, a new solubility curve with the corrected Cs was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the Cs and Cp of the solution.


Microbial Reduction and Quality Changes in Powdered White and Black Pepper by Treatment with Compressed Oxygen or Carbon Dioxide Gas

Powdered white and black peppers were treated with high-pressure oxygen (10 MPa) or carbon dioxide (5 MPa)at 70 _ 100 to decrease the microbial populations. The degree of microbial reduction in the pepper after treatment with high-pressure gases was dependent on the temperature and duration of treatment. Mesophilic aerobic bacterial counts of less than 103 CFU/g in white and black pepper were achieved with gas pressurization at 100 for 10 and 40 min, respectively. The reduction in the microbial population of the two types of pepper was attributed to the microbicidal effects of high-pressure gas and heat treatment. The piperine content of the pepper treated with gases decreased by approximately 10%. However, treated pepper also showed lower L* values, indicating a darker color, and qualitative alterations in volatile compounds.


Identification of triangular-shaped defects often appeared in hard-sphere crystals grown on a square pattern under gravity by Monte Carlo simulations

In thispaper,we have successfully identified the triangular-shaped defect structures withstackingfault tetrahedra. These structure often appeared in hard-sphere (HS) crystals grown on a square patter nunder gravity. Wehave, so far, performed Monte Carlosimulations of the HS crystals under gravity. Single stacking faults as observed previously in the HScrystals grown on a flat wall were not seen in the case of square template. Instead, defect structures with triangular appearance in xz- and yz- projections were appreciable. We have identified them by looking layer by layer. Those structures are surrounded by stacking faults along face-centered cubic (fcc) {111}. Also, we see isolated vacanciesandvacancy interstitial pairs, and we have found octahedral structures surrounded by stacking faults along fcc {111}.


Crystallization of Brownian particles in thin systems constrained by walls

Physical Review E, Statistical, Nonlinear, and Soft Matter Physics


Dislocations in High-quality Glucose Isomerase Crystals Grown from Seed Crystals


Ordering of Brownian Particles from Walls Due to an External Force

Keeping the formation of colloidal crystal under a centrifugal force in mind, we study the ordering of Brownian particles under a uniform external force. Owing to the external force, the particles move in the direction of the external force. Near walls, the density of particles increases and the ordering of particles occurs on the walls at first. Then, the ordering in bulk proceeds gradually. Domains with the face-centered cubic and hexagonal close-packed structures are created in bulk. By controlling the direction and strength of external force, the ratio of the two types of structures changes. © 2014 Elsevier B.V. All rights reserved.


Colloidal crystallization on tilted substrates under gravitational fields

We show usefulness of a side wall of a tilted growth container during continuous growth of colloidal crystals under gravitational fields. The side wall works as an effective substrate which enables both enlargement of grains of colloidal crystals and control of crystallographic orientations of the grains. We successfully fabricated large grains of polystyrene face-centered cubic colloidal crystals which align with their {111} planes parallel to a side wall by tilting a growth container during centrifugation. Although, without tilting a growth container, widths and crystallographic orientations of grains remained unchanged once the widths become asymptotic values, we could increase both widths of grains and population of the grains which aligned with their {111} planes parallel to a side wall with terminating grains of the other orientations by tilting the container.


Effect of container shape and walls on solidification of Brownian particles in a narrow system

We carry out Brownian dynamics simulations and study the ordering of particles under a uniform external force in a narrow system. In our previous studies [M. Sato et al. Phys. Rev. E 87, 032403 (2013); J. Phys. Soc. Jpn. 82, 084804 (2013)], we showed that the ordering of particles depends on the direction of the external force. In the studies, however, the system size and the number of particles are small, so that the behaviors we observed corresponds to the motions in the initial stage of crystallization. In this paper, using a longer container and more particles, we investigate how solidification in a narrow system proceeds. We also study the effect of the shape of simulation box on the ordering of particles. When we use a rhomboid as a simulation box, the ratio of the particles with the face-centered cubic structure to those with the hexagonal close-packed structure is larger than that in a cuboid system.


Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry

The growth rate vs. supersaturation of a lysozyme crystal was successfully measured in situ together with the crystal surface observation and the concentration measurements onboard the International Space Station. A Michelson-type interferometer and a Mach-Zehnder interferometer were, respectively, employed for real-time growth rate measurements and concentration field measurements. The hardware development, sample preparation, operation, and analysis methods are described.


Colloidal crystallization utilizing interfaces of unidirectionally growing ice crystals


Crystallization of Brownian Particles from Walls Induced by a Uniform External Force

Keeping the formation of colloidal crystal under a centrifugal force in mind, we study the ordering of Brownian particles induced by a uniform external force. When the uniform external force is added, the particles move in the direction of the external force and the density of particles near walls becomes high. The ordering of particles starts on the walls, and successive ordering in bulk occurs near the walls. In bulk, both domains of the face-centered cubic structure and hexagonal close-packed structure appear. By controlling the direction and the strength of the external force, the number of ordered particles and distribution of cluster size are changed.

Journal of the Physical Society of Japan


Vanishing linear term in chemical potential difference in volume term of work of critical nucleus formation for phase transition without volume change

A question is given on the form n(μ_β-μ_α) for the volume term of work of formation of critical nucleus. Here, n is the number of molecule undergone the phase transition, μ denotes the chemical potential, α and β represent the parent and nucleating phases, respectively. In this paper we concentrate phase transition without volume change. We have calculated the volume term in terms of the chemical potential difference μ_{re}-μ_{eq} for this case. Here, μ_{re}$ is the chemical potential of the reservoir and μ_{eq} that at the phase transition. We have W_{vol} = -[(κ_β-κ_α)/2v_{eq}^2] (μ_{re}-μ_{eq})^2 V_βwith κ denoting the isothermal compressibility, v_{eq} being the molecular volume at the phase transition, V_β the volume of the nucleus.


Grand potential formalism of interfacial thermodynamics for critical nucleus

In the nucleation theory, the work of formation of a nucleus is often denoted by W=ΔG. This convention misleads that the nucleation should be considered in the isothermal-isobaric system. However, the pressure in the system with a nucleus is no longer uniform due to Laplace's equation.Instead, the chemical potential is uniform throughout the system for the critical nucleus. Therefore, one can consider the nucleation in the grand ensemble properly. Accordingly, W is found to be the grand potential difference and the interfacial tension is also turned to be an interfacial excess grand potential. This treatment is not entirely new; however, to explicitly treat in the grand potential formalism is for the first time. We have successfully given an overwhelmingly clear description.


Formation of a crystal of Brownian particles under a uniform external force

To keep the formation of colloidal crystal under a centrifugation in mind, we study ordering of Brownian particles under a uniform external force. When the force is added to Brownian particles distributing uniformly in the system, the particles drift and the density of particles near walls increases. Ordering of particles on the walls occurs at first and ordering in bulk occurs in succession. In bulk, both the clusters with face-centered cubic structure and those with the hexagonal close-packed structure appear. The distribution of cluster sizes changes with the direction of external force.

Physical Review E, Statistical, Nonlinear, and Soft Matter Physics


Gravitational Annealing of Colloidal Crystals

A silica colloidal crystal obtained by centrifugation at 9 G for 2 days in water was annealed by additional stronger centrifugation at 50 G for 5 days. The number of the striations observed in the colloidal crystal under crossed polarized light decreased at some parts in a growth container after the additional centrifugation, while the number also increased at the other parts. The decrease probably shows the shrinkage of the stacking disorders under high gravity, while the increase probably shows the production of new stacking disorders.


Monte Carlo Simulation of Defects in Hard-Sphere Crystal Grown on a Square Pattern

Monte Carlo simulations of hard-sphere (HS) crystal gown on a square patterned wall under gravity have been performed. While previous simulations were performed with step-wise controlled gravity, in the present simulations the gravity which is constant from the first has been applied. In the case in which a flat wall is used as the bottom wall, if a large gravity is suddenly applied, the system does polycrystallize. On the other hand, in the present simulations, despite the sudden application of gravity, the system has not polycrystallize. Crystalline nucleation on the square pattern and successive crystal growth upward are suggested to overcome the homogeneous nucleation inside and result in. Defect disappearance, which has been essentially the same as that for the case with step-wise controlled gravity, has also observed for the present case. The characteristic of the square patterned bottom wall simulation with a large horizontal system size has been existence of triangular defects suggesting stacking tetrahedra.


Solubility measurements by in situ observation of the apex region formed by the (110), (1-10) and (101) faces of tetragonal lysozyme crystals


Effects of high pressure on the step velocity on the {110} faces of tetragonal lysozyme crystals


Effects of gas pressurization with ethylene on the ultrastructure of the yeast Saccharomyces cerevisiae

We investigated ultrastructural changes in the yeast Saccharomyces cerevisiae when exposed to compressed ethylene gas. Transmission electron microscopy (TEM) revealed that intracellular organelles in yeast cells treated with compressed ethylene at up to 0.640 MPa (6.4 atm), especially the nuclear and plasma membranes, were seriously damaged.

Bioscience, Biotechnology, and Biochemistry


Precise characterization of grain structures, stacking disorders and lattice disorders of a close-packed colloidal crystal

The perpendicular fracture surface of a dried colloidal crystal with pillar-like grains, obtained by centrifugation of a dispersion of polystyrene particles, was observed using a scanning electron microscope. Many grain boundaries on the fracture surface were observed at the particle level. Most of the particles on the surface showed a face-centered cubic (FCC) array. Although some grains were single FCC ones, other FCC grains contained some stacking disorders. Most of the surface was covered with such grains, and the grain boundaries formed a mosaic-like pattern. From these results, we confirmed that the colloidal crystals obtained by centrifugation formed a bundle structure of pillar-like FCC grains. A fracture surface adjacent to the side wall of the growth cell was also observed. The surface was composed of several layers. In the uppermost layer closest to the wall, numerous point defects and mismatches of triangular lattices between the neighboring two-dimensional islands were observed. These mismatches and point defects probably generated several lattice defects in the crystal. Similar generation of lattice defects probably occurred at the bottom of the container or the growth front of the crystals. Screw dislocations were also found in the layers, although they were not observed frequently. From these results, it was concluded that two-dimensional nucleation growth and spiral growth probably occurred on the crystal-dispersion interfaces of colloidal crystals as well as on the surface of atomic crystals.


Effects of temperature, pressure, and pH on the solubility of triclinic lysozyme crystals

We measured the temperature dependence of the solubility, Ce, of triclinic lysozyme crystals on temperature at two pressures (0.1 and 100 MPa) and five pHs (4.3, 4.4, 4.5, 4.6, 4.7) by in situ observation of the morphological changes in the crystals. The solubility increased with increasing temperature and pressure. The enthalpies of dissolution (ΔH) decreased with increasing pressure when we used the same 50 mM sodium acetate buffer solution (pH = 4.5 at 0.1 MPa). The effects of pH on solubility were somehow complicated. Although the solubilities at pH 4.7 did not differ from those at pH 4.5, those at pH 4.3 were significantly larger than those at pH 4.5. Since the pH of an acetate buffer is known to decrease from 4.71 to 4.52 at 98.1 MPa and 25 ºC [El'yanov et al. Aust. J. Chem. 28 (1975) 933], the pH of the acetate buffer (pH 4.5 at 0.1 MPa) decreased to pH ~ 4.3 as pressure increased to 100 MPa. Thus, from the viewpoint of ``constant pH'', ΔH increased with increasing pressure, since ΔH at pH 4.3 and 0.1 MPa was smaller than that at 100 MPa. This inconsistency is probably due to the change in the hydration state in the crystal and solution with decreasing pH (from 4.5 to 4.3).


Colloidal crystallization by a centrifugation method

Colloidal crystals of high particle concentration were smoothly and continuously fabricated by simple centrifugation of a dispersion of polystyrene particles in water. The larger the applied centrifugal acceleration at the bottom of the growing crystals (i.e., the interior wall of the container, the higher the growth rates became. The average grain sizes in the crystals increased with decreasing , and below a critical value of , we obtained relatively large columnar-shaped grains. The average volume fraction of the obtained colloidal crystals ave. increased with an increase in the dispersion's KCl concentration CKCl: at = 82 G, ave. = 0.57 and 0.61 at CKCl = 2 × 10-3 and 8 × 10-3 molkg-1 water, respectively. The grain size also increased with an increase in CKCl. The maximum grain size in this study was greater than 0.3 mm in width and 8 mm in length.


Activation volume of crystallization and effects of pressure on the three-dimensional nucleation rate of glucose isomerase

An activation volume, DV , of the solute incorporation process at the kink site of a glucose isomerase (GI) crystal was estimated using the pressure dependency of the step kinetic coefficient, betastep, and the three-dimensional nucleation rates J of GI crystals were analyzed using classical nucleation theory. DV took a negative value. The sticking parameter, s, for the addition of a GI tetramer to a critical nucleus drastically increased with increasing pressure. This was probably due to the decrease in the activation energy needed for the addition of the GI tetramer.


Effects of compressed hydrocarbon gases on the growth activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane < ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.


Design of a stand-alone-type Beryllium vessel for high-pressure protein crystallography

A standalone-type beryllium (Be) high-pressure crystallography vessel has been developed. Using a coupler-joint unit and a pressure valve, we could keep the pressure in the vessel constant at 100+/-1 MPa for more than 24 h without connecting to a pressure-generating apparatus. Diffraction spots of a glucose isomerase (GI) crystal under 100 MPa were collected using the vessel and a rotating copper-anode in-house x-ray generator (0.8 kW). We successfully collected a 2.0 A resolution data set of a 0.5 mm size GI crystal in an aqueous solution at 100 MPa.


Interplay between elastic fields due to gravity and a partial dislocation for a hard-sphere crystal coherently grown under gravity: driving force for defect disappearance

We previously observed that an intrinsic staking fault shrunk through a glide of a Shockley partial dislocation terminating its lower end in a hard-sphere crystal under gravity coherently grown in <001> by Monte Carlo simulations [Mori et al., Molec. Phys. 105, 1377 (2007)]; it was an answer to a one-decade long standing question why the stacking disorder in colloidal crystals reduced under gravity [Zhu et al., Nature 387, 883 (1997)]. Here, we present an elastic energy calculation; in addition to the self-energy of the partial dislocation [Mori et al., Prog. Theor. Phys. Suppl. 178, 33 (2009)] we calculate the cross-coupling term between elastic field due to gravity and that due to a Shockley partial dislocation. The cross term is an increasing function of the linear dimension R over which the elastic field expands, showing that a driving force arises for the partial dislocation moving toward the upper boundary of a grain.


Effects of compressed unsaturated hydrocarbon gases on yeast growth

The effect of compressed unsaturated hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated by microcalorimetry. The growth thermograms showed that unsaturated hydrocarbon gases inhibited yeast growth. As an approach to determining the comparative toxicity of unsaturated hydrocarbon gases, we determined the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP). On the basis of the IP(50) and MIP values, the inhibitory potency of the gases increased in the order ethylene < propylene < 1-butene. Additionally, scanning electron microscopy showed that cells treated with unsaturated hydrocarbon gases were damaged, including invagination of the cell surface.

Annals of the New York Academy of Sciences


First Direct Observation of Elementary Steps on the Surfaces of Glucose Isomerase Crystals under High Pressure


Disappearance of a Stacking Fault in Hard-Sphere Crystals under Gravity

In the first part of this paper, a review is given on the mechanism for the disappearance of an intrinsic stacking fault in a hard-sphere (HS) crystal under gravity, which we recently discovered by Monte Carlo (MC) simulations [A. Mori et al., J. Chem. Phys. 124 (2006), 17450; Mol. Phys. 105 (2007), 1377]. We have observed, in the case of fcc (001) stacking, that the intrinsic stacking fault running along an oblique direction shrunk through the gliding of a Shockley partial dislocation at the lower end of the stacking fault. In order to address the shortcomings and approximations of previous simulations, such as the use of periodic boundary condition (PBC) and the fact that the fcc (001) stacking had been realized by the stress from the small PBC box, we present an elastic strain energy calculation for an infinite system and a MC simulation result for HSs in a pyramidal pit under gravity. In particular, the geometry of the latter has already been tested experimentally [S. Matsuo et al., Appl. Phys. Lett. 82 (2003), 4283]. The advantage of using a pyramidal pit as a template as well as the feasibility of the mechanism we describe is demonstrated.

Progress of Theoretical Physics Supplement


Development of the Food Sterilization Equipment by Oxygen-Nitrogen Gas Hybrid Pressurization System

酸素・窒素ガスハイブリッド加圧食品殺菌装置の開発

Microbial spoilage, including fermentation and oxidation is particularly an important factor that influences the quality of foods. In previous studies we showed that sudachi (sour citrus fruit) juice could be sterilized with oxygen gas pressurization at 10 MPa and 50°C without loss of its flavor and color. Furthermore, we reported a method to remove the dissolved oxygen in sudachi juice with nitrogen gas pressurization. However, all these results were obtained using a small-scale batch process (500 ml or less in capacity). In this research, we designed a new commercial scale food processing apparatus for the sudachi juice based on the above findings. This device consists of two parts: a sterilization part with oxygen gas pressurization and a dissolved oxygen removal part with nitrogen gas pressurization, and it has a big advantage that a large amount of sudachi juice can be treated continuously at the flow rates up to 40 L/h. Using this apparatus, the sudachi juice was sterilized under similar operation conditions (50°C and 10 MPa for 1 minute) to those of the small-scale batch system. Although the performance of dissolved oxygen removal and the quality conservation of the juice (such as flavor and color) is under investigation now, the facility and production costs are expected to be apparently less than those of high-pressure processing (liquid pressure). Because, our treatments can be completed in a short time at relatively low pressures using “oxygen-nitrogen hybrid pressurization system”. We consider further improvements of the apparatus and hope that the practical application of the method can be realized in food industry in the near future.

我々の研究から，スダチ果汁を100気圧程度の酸素ガスで直接加圧すると，香りや色等の品質を保持したまま，果汁中の酵母を殺菌でき，さらに，窒素ガスで加圧処理を行うと，果汁中の溶存酸素を除去できることが分かった．これらの研究成果は500 ml以下のバッチ式加圧処理装置によるものであったが，今回，装置のスケールアップと果汁の連続処理を実現し，高品質の柑橘系「生」果汁を生産する，一連のハイブリッドシステム装置を設計・試作した．また，試作した"酸素・窒素ガスハイブリッド加圧食品殺菌装置"で，スダチ果汁を処理したところ，基礎研究と同じ，50°C，10 MPa，1分間の処理で，果汁中の酵母を殺菌することができた．

High Pressure Bioscience and Biotechnology

高圧バイオサイエンスとバイオテクノロジー


Calorimetric Studies on the Growth Behavior of Yeast under Some Compressed Hydrocarbon Gases

代謝熱測定法による炭化水素ガス加圧下の酵母の増殖挙動解析

In this study, the inhibitory effect under compressed hydrocarbon gases on yeast growth was investigated quantitatively by microcalorimetry. The growth thermograms (heat output - incubation time curve) were obtained during incubation of yeast under various compressed hydrocarbon gases. When the gas pressure increased, the curves of thermograms shifted to the right, indicating an obvious inhibitory action on the yeast growth. After quantification of growth inhibition at various pressures, we determined the 50% inhibitory pressure (IP50) and the minimum inhibitory pressure (MIP) values as indices which represent toxic potency of each gas. The lower the IP50 and MIP values, the greater the growth inhibitory effects of the gas. Based on these values, the inhibitory potency of the gases increased in the order: methane < ethane < propane < i-butane < n-butane. In addition, the toxicity of some hydrocarbon gases was found to be correlated to their hydrophobicity. These results suggest that hydrocarbon gases interact with some hydrophobic region of the cell membrane, resulting in modification of membrane structure and function. However, no evidence was obtained in the yeast treated with compressed hydrocarbon gases as to the membrane damage. Therefore, we investigated the effects of hydrocarbon gases on the leakage of 260 nm absorbing material from treated cells. The release amount of 260 nm absorbing material increased with increasing pressure. This indicates that hydrocarbon gases affect the plasma membrane, particularly the nuclear membrane. Furthermore, we examined the effects of hydrocarbon gases on the morphology of the yeasts by scanning electron microscopy. Scanning electron microscopy showed that yeasts when exposed to compressed hydrocarbon gases undergo a dramatic morphological change that includes invagination of the cell surface.

一連の炭化水素ガス(C1∼C4)加圧下で，代謝熱測定法により酵母の増殖挙動をモニタリング・解析し，その細胞毒性を定量的に評価した．各気体ごとに，50%増殖阻止圧力(IP50: Inhibitory pressure 50)と最小増殖阻止圧力(MIP: Minimum inhibitory pressure)を決定した．その結果，炭化水素ガスの種類，異性体によって毒性が異なり，n-ブタン>i-ブタン>プロパン>エタン>メタンの順に毒性が大きいことが明らかになった．炭化水素ガスの毒性は，その疎水性との間に強い相関関係があったことから，炭化水素ガスが酵母の細胞膜などの疎水性領域と作用し，生体膜に何らかの影響を与えているものと考えられる．また，炭化水素ガス加圧により，酵母から核酸関連物質の漏出が確認された．さらに，炭化水素ガス加圧による酵母の形態変化を走査電子顕微鏡により観察したところ，細胞が変形，陥没している様子が認められた．

高圧バイオサイエンスとバイオテクノロジー


Succession of stacking fault in hard-sphere crystal under gravity by Monte Carlo simulation

We have performed Monte Carlo simulations of hard spheres under gravity. Vertical boundaries are hard walls, which are well separated with each other. On the other hand, the periodic boundary condition is imposed in the horizontal direction. While we previously reported enhancement of crystallinity as well as crystallization due to gravity, we present here the results that demonstrate the succession of a defect. In case that the crystal formed at the bottom of the system includes kinds of stacking disorders for the (001) growth, twin band structure develops as mediated by a stacking disorder succeeded in the crystal formed in the fluid region which lies on the bottom crystal. In case that the stacking structure along horizontal direction changes from the (111) stacking to the (001) stacking, twin band structure in the (001) stacking region develops as succeeded in the crystal transformed. The twin band structure also becomes large with its upward growth.


Effects of Some Compresed Hydrocarbon Gases on Yeast Growth

High Pressure Bioscience and Biotechnology


Effects of Antioxidants (α-Lipoic Acid and Vitamin E) on the Phase Transition Temperature of Bilayer Membrane of Phospholipids under Oxygen-Gas Pressure


Effects of Some Compressed Rare Gases of Yeast Growth


Sterilization of Sudachi Juice by Hybrid Gas Compression of Oxygen and Nitrogen


Shrinking stacking fault through glide of the Shockley partial dislocation in hard-sphere crystal under gravity

Disappearance of a stacking fault in the hard-sphere crystal under gravity, such as reported by Zhu et al. [Nature 387 (1997) 883], has successfully been demonstrated by Monte Carlo simulations. We previously found that a less ordered (or defective) crystal formed above a bottom ordered crystal under stepwise controlled gravity [Mori et al., J. Chem. Phys. 124 (2006) 174507]. A defect in the upper defective region has been identified with a stacking fault for the (001) growth. We have looked at the shrinking of a stacking fault mediated by the motion of the Shockley partial dislocation; the Shockley partial dislocation terminating the lower end of the stacking fault glides. In addition, the presence of crystal strain, which cooperates with gravity to reduce stacking faults, has been observed.


Colloidal crystallization by centrifugation

遠心沈降法を用いたコロイド結晶の作製

Columnar-shaped colloidal crystals (width and length of the columns -0.1-1.0 mm and 10 mm, respectively) have been grown by centrifugation of the dispersion of polystyrene particles. The crystals were re-dispersed reversibly. Close-packed colloidal crystals were obtained by drying the columnar crystals gently. The dried crystals contained no macroscopic cracks over several mm3. Some stacking disorders were observed on the cross sections of the dried crystals. The average grain size of the columnar crystals increased with decreasing centrifugal acceleration. Too fast centrifugation caused the sedimentation of randomly oriented small grains in front of the growth interfaces.

三次元的な広がりをもったグレイン(結晶粒)をもつ密充填のコロイド結晶を制御して効率よく作製するため，比重の小さなポリスチレン粒子を遠心沈降濃縮した．その結果，0.1∼1.0 mm の幅で 10 mm 程度の長さをもつ柱状のグレインの集合体からなる高粒子密度のコロイド結晶を 84G(1G=9.8 ms-1)の遠心加速度により，従来の自然沈降法よりも短い時間(1週間)で作製できた．遠心機から取り出した結晶は，単に逆さま(固液界面で分散液のほうに向かって 1G の加速度がかかる状態)にすることで可逆的に溶解したことから，通常の結晶の高品質化技術を適用できる可能性が示唆された．また，遠心沈降法によって得られた高粒子密度(体積分率φ∼0.4)のコロイド結晶をゆっくり乾燥させることで，結晶構造を壊すことなく密充填の乾燥コロイド結晶を得ることができた．遠心沈降法によって得られるコロイド結晶の平均的なグレインサイズは，沈降速度を減少させることによって大きくなった．これは，沈降速度の減少により，セル底面において生成した結晶核が減少したことによると考えられる．

Japanese Journal of Polymer Science and Technology


Solubility Measurements of Tetragonal Lysozyme Crystals

International Journal of Modern Physics B


Monte Carlo simulation of crystal-fluid coexistence states in the hard-sphere system under gravity with step-wise control

Monte Carlo (MC) simulations were performed for hard spheres (with diameter sigma and mass m) placed between well-separated upper and lower hard walls. A periodic boundary condition was imposed in the horizontal direction. The system was exposed to the gravitational field with the acceleration due to gravity g. After preparing a melt as the initial state, g was increased stepwise up to mgsigma/kBT([equivalent]g*)=1.5 or 2.0 with an increment Deltag*=0.1; kBT is the temperature multiplied by Boltzmann's constant. We maintained g* at each value for 2.0×105 MC cycles. The transition of the system into a metastable state such as a polycrystalline state due to trapping phenomena was successfully avoided. A monotonic increase and subsequent saturation were observed for the development of the crystalline region formed at the bottom of the system. The development of this region accompanied a shrinkage of the defective (or less ordered) crystalline region that was formed between the bottom region and the fluid phase. As the development of the bottom region almost saturated, the defective region grew upward again.


Crystal structure of hard spheres under gravity by Monte Carlo simulation

Monte Carlo simulations were performed for hard spheres (HSs) under gravity. The gravity was increased stepwise. HSs were placed between the bottom and the top hard walls. For g*R0.9, we observed that a `sediment' was comprised of two crystalline and one fluid regions. Here, g* is defined by g*Zmgs/kBT with m being the mass of a particle, s the HS diameter, g the acceleration due to gravity, and kBT the temperature multiplied by Boltzmann's constant. The bottom crystal was less defective or well-ordered and the crystal lay between the bottom one and the fluid phase was defective or less-ordered. In this paper, we investigate the structure of the crystals. Despite no apparent defects, the crystal has highly been distorted. That is, the fcc lattice has been contracted in the vertical direction more than in the horizontal direction. The crystal fluid coexistence condition for the bulk HS system does, in principle, not hold for the present systems at the crystal fluid interface. In addition, though the fine scale density profile exhibits a discontinuity apparently across the crystal crystal interface, the interlayer separation increases linearly with the height.

Science and Technology of Advanced Materials


The biological effects of high-pressure gas on the yeast transcriptome

The aim of the present study was to examine the feasibility of DNA microarray technology in an attempt to construct an evaluation system for determining gas toxicity using high-pressure conditions, as it is well known that pressure increases the concentration of a gas. As a first step, we used yeast (Saccharomyces cerevisiae) as the indicator organism and analyzed the mRNA expression profiles after exposure of yeast cells to nitrogen gas. Nitrogen gas was selected as a negative control since this gas has low toxicity. Yeast DNA microarray analysis revealed induction of genes whose products were localized to the membranes, and of genes that are involved in or contribute to energy production. Furthermore, we found that nitrogen gas significantly affected the transport system in the cells. Interestingly, nitrogen gas also resulted in induction of cold-shock responsive genes. These results suggest the possibility of applying yeast DNA microarray to gas bioassays up to 40 MPa. We therefore think that "bioassays" are ideal for use in environmental control and protection studies.

Brazilian Journal of Medical and Biological Research


Enhancement of Crystallization of Hard Spheres by Gravity: Monte Carlo Simulation

We present the effects of gravity on the growth of a hard-sphere (HS) crystal as determined by Monte Carlo simulations. HSs were confined between hard walls at the top (z . Lz) and the bottom (z . 0) of the system with a periodic boundary condition in the horizontal direction. After preparing a melt state as an initial state, the gravity was suddenly switched on. The values of the gravity were mg_=kBTd_ g_T . 0:1; 0:2; . . . ; 2:0, where m was the mass of a HS, g the acceleration of gravity, _ the HS diameter, and kBT the temperature multiplied by Boltzmann's constant. We observed the enhancement of crystallization due to gravity up to g_ . 0:7 and that the crystals of largest size were formed at g_ . 0:7{0:9. On the other hand, for g_ _ 1:0, the top position of the HS crystal that was grown from the bottom became lower with an increase in the gravity, where crystals with various axis directions coexisted. The polycrystallization is speculated to occur due to polynucleation. These results are qualitatively consistent with those of an experimental study of the centrifugal sedimentation of colloidal crystallization.

Japanese Journal of Applied Physics, Part 1 (Regular Papers & Short Notes)


Effect of high-pressure gas on yeast growth

Microcalorimetry is a useful tool for monitoring the growth behavior of microorganisms. In this study, microcalorimetry was used to investigate the effects of nitrogen, air, oxygen, nitrous oxide, argon, and krypton at high pressure on the growth of the yeast Saccharomyces cerevisiae. Growth thermograms (metabolic heat vs. incubation time) were generated to estimate metabolic activity under compressed gases and to determine the 50% inhibitory pressure (IP(50)) and minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of compressed gases. Based on MIP values, the most toxic to the least toxic gases were found to be: O(2) > N(2)O > air > Kr > N(2) > Ar.

Bioscience, Biotechnology, and Biochemistry


Removal of Dissolved Oxygen in Sudachi Juice by Nitrogen Gas Pressurization

窒素ガス加圧によるスダチ果汁中溶存酸素の除去

果汁中の溶存酸素は，さまざまな形で品質劣化の原因となる．本研究は，果汁を窒素ガスで加圧したのち減圧することで，窒素が溶存酸素を同伴する作用を利用して酸素脱気する技術に関するものである．20秒間隔で，加圧と除圧を7回繰り返すことにより，効率的に脱気することができた．30℃，10 MPaで処理することにより，溶存酸素量を0.02 mg/lまで下げることができた．本技術はこれまでにない新しい技術であり，特許の出願につながった．


High Pressure Acceleration of the Growth Kinetics of Glucose Isomerase Crystals

グルコースイソメラーゼ結晶の{101}面の成長·溶解速度を0.1, 100 MPaでその場観察により測定した．25℃において，溶解度は0.1 MPaでCe = 3.1 ± 0.9 mgml-1，100 MPaで 2.6 ± 0.5 mg ml-1 となった．同じ2.5という過飽和度で比較した結果，成長速度は100MPaにおいて0.1MPaの7.6倍になった．この結果から，加圧によって結晶の成長速度が速度論的に加速されることが初めて明らかになった．二次元核成長モデルを用いて解析した結果，加圧による成長速度の加速の原因は，主に表面自由エネルギーの減少にあることが分かった．


Sterilization of Sudachi Juice by Compressed Oxygen Gas

スダチ果汁の酸素ガス加圧殺菌

新しい技術である酸素ガス加圧殺菌に関する研究．スダチ果汁を用い，果汁中酵母(Candida boidinii SYM-1)に対する加圧酸素ガスの毒性を調べ，殺菌条件を見積もった．30℃+5.0 MPaで15 min，40℃+10.0 MPaでは5 min，そして50℃+5.0 MPaでは1 min未満で殺菌が可能であった．50℃では，ビタミンCの減少を防ぐことができた．従来の加熱殺菌では，品質の劣化が著しかったが，本法により品質を維持しながら殺菌が可能となった．本研究は1件の特許出願につながった．


Effects of high pressure on the solubility and growth kinetics of monoclinic lysozyme crystals

単斜晶系リゾチーム結晶の(010)面と(0-10)面の平均成長速度R<010>を0.1, 100MPaの圧力下でその場観察により測定した．成長速度の濃度依存性から溶解度を求めた結果，溶解度は圧力の増加に伴い増加した．0.1, 100MPaにおいて同じ過飽和度下で結晶の成長速度を比較した結果，加圧に伴い，結晶の成長速度は増加した．二次元核成長モデルを用いて解析した結果，その原因は，表面自由エネルギー，活性化エネルギー，そしてステップ上のキンク間距離の加圧に伴う減少にあることがわかった．


Bioassay of various pesticides by microcalorimetry measuring the metabolic heat of yeast

Using a microcalorimeter, the heat evolved during incubation of yeast cultures at 30 degrees C was detected in the form of a growth thermogram (metabolic heat-incubation time curve). The correlation between the growth thermograms and the growth curves obtained by colony counting or from the turbidity measurements of the cultures was very good. In this study, the effects of 12 pesticides that are used widely on golf courses on the growth thermograms of yeast were investigated and the inhibitory action of the pesticides on the growth of yeast was quantitatively evaluated using a microcalorimeter (Biothermo Analyzer). The growth thermograms were applied to estimate the microbial activity of yeast in the presence of pesticide and to determine the 50% inhibitory concentration (K) and minimum inhibitory concentration (MIC), which can be regarded as good indexes of the toxic potency of pesticides. It was found that the addition of TPN, captan, thiuram and copper oxide to the yeast cultures clearly induced inhibitory action on the growth of yeast cells. On the other hand, the other 8 pesticides, iprodione, isoprothiorane, chloroneb, pencycuron, metalaxyl, toleclofos-methyl, flutolanil, and mepronil did not affect the growth over the concentration range up to 30 mg/l.


Effects of high pressure on the growth kinetics of orthorhombic lysozyme crystals

斜方晶系リゾチーム結晶の(011)面と(0-11)面の成長過程を0.1, 100 MPaでその場観察した．0.1と100MPaで同じ過飽和度で比較したところ，両面の成長速度は加圧に伴い減少することが分かった．二次元核成長モデルで解析した結果，加圧に伴う成長速度の減少は溶質分子の結晶への取込み過程における活性化エネルギーの増加によって説明できることがわかった．


Effects of dissolved gases on the phase transition of liposomal bilayer membranes

リン脂質二分子膜の相転移に及ぼす溶存気体の影響

2種類のリン脂質二分子膜(DMPC,DPPC)のゲル-液晶間の相転移温度(Tm)を，様々な気体と接触させた開放系で加圧して決定した．使用した気体は窒素，酸素，水素，ヘリウム，アルゴンの5種類の単一気体と4種類の混合気体である．Tmに対する圧力の影響は，加圧によりTmが直線的に上昇する気体のグループと低下するグループの2つに分かれた．加圧によりTmが低下するグループの気体は，水に対する溶解度が大きいものが多いことから，溶存気体の濃度が転移温度の低下に関与していることが推測された．特に亜酸化窒素や酸素，アルゴンなどが，Tmを押し下げる効果が大きく，これらの溶存気体のリン脂質二分子膜骨格に対する作用の大きさが示唆された．


Solubility of α-amino acids in water under high pressure - glycine, L-alanine, L-valine, L-leucine, and L-isoleucine

glycine, L-alanine, L-valine, L-leucine, and L-isoleucineの水に対する溶解度を298.15Kで400MPaの圧力まで測定した．その結果，アミノ酸の種類によって様々な結果が明らかになった．glycineは加圧にともない溶解度が減少したのに対し，L-alanineの溶解度は逆に増加した．L-valineとL-isoleucine は100MPa付近に溶解度の極大があり，L-leucineは200MPa付近に固相相転移が起こっていることが明らかになった．


リン脂質二分子膜に及ぼす溶存気体の影響

リン脂質二分子膜に及ぼす溶存気体の影響

ジミリストイルフォスファチジルコリン二分子膜のゲル-液晶主転移温度(Tm)を様々な気体と接触させた解放系で加圧して決定した．使用した気体は酸素，窒素，水素，アルゴンの4種類の単一気体と空気，20.0%酸素(ヘリウムベース)，10.1%亜酸化窒素(窒素ベース)及び4.69%水素(窒素ベース)の4種類の混合気体である．Tmに対する圧力の影響は加圧によりTmが直線的に上昇する気体のグループと低下するグループの2つに分かれた．加圧によりTmが低下するグループの気体は，水に対する溶解度が大きいものが多いことから，溶存気体の濃度が転移温度の低下に関与していることが推察された．特に亜酸化窒素(笑気ガス)や酸素が，Tmを押し下げる効果が大きく，これらの溶存気体のリン脂質二分子膜骨格に対する作用の大きさが示唆された．


Effects of Pressure and Temperature on the Solubility of Monosodium l-Glutamate Monohydrate in Water

グルタミン酸ナトリウム一水和物の水に対する溶解度を0.10-300 MPaの圧力範囲で298.15 Kで測定した．高濃度水溶液の密度と，飽和溶液の溶解熱についても常圧下で測定した．溶解度msは加圧に伴い増加し，その圧力係数dlnms/dp=(2.0±0.1)X10^(-10) Pa^(-1)は，密度測定によって求められた部分モル体積と活量係数と結晶のモル体積から熱力学的に求められた値(2.1±0.2)X10^(-10) Pa^(-1)と一致した．溶解熱から求められた温度係数dlnms/d(1/T)は，Ogawaらによる溶解度の温度係数と良く一致した．


アルコールショック酵母の増殖とストレス応答

アルコールショック酵母の増殖とストレス応答

酵母に一連のアルコール(メタノール，エタノール，1-プロパノール，2-プロパノール，1-ブタノール，2-ブタノール，iso-ブタノール，tert-ブタノール)を短時間接触させることでケミカルショックを加え，誘発される二次的なストレスに対する耐性(熱耐性と圧力耐性)を見積もった．また，マイクロ熱量計を用い，培地に同様なアルコールを添加した酵母の増殖サーモグラムを描き，増殖活性に及ぼすアルコールの影響を調べた．耐性実験，サーモグラム実験共にアルコールの性質により大きく影響を受けることが分かった．アルコールの分子構造や，熱測定で求められる増殖速度定数と最小増殖阻止濃度(MIC)などをもとに，酵母細胞に及ぼす一連のアルコールの影響を調べた．一連のアルコールについて，酵母に対するMICとオタマジャクシに対する麻酔作用のED50の間には良い直線関係がみられた．


Effects of pressure on growth kinetics of tetragonal lysozyme crystals

高圧力下における正方晶系リゾチーム結晶の成長kineticsをその場測定により研究した．{110}面と{101}面の成長速度を0.1, 50, 100 MPaで測定した．両方の面とも，同じ過飽和度で比較した時，加圧により成長速度が減少した．二次元核成長のbirth and spread modelでfittingしたところ，平均ステップレッジ表面自由エネルギーが加圧とともに増加することが，成長速度の減少につながるということが明らかになった．


Solubility of tetragonal and orthorhombic lysozyme crystals under high pressure

マイケルソン干渉計を用いた溶解度の迅速測定法を高圧力下のリゾチームの溶解度測定に適用した．正方晶系リゾチームに関しては0.1, 50, 100 MPa，斜方晶系リゾチームに関しては，0.1, 100 MPaで温度の関数として溶解度を測定した．その結果，加圧に伴い，正方晶系の方は溶解度が増加し，それに対して斜方晶系では減少した．双方とも溶解エンタルピーは加圧に伴い減少した．


In situ measurements of the solubility of crystals under high pressure by an interferometric method

マッハツェンダー干渉計と光学窓付きの圧力容器を用いて，高圧力下での結晶の溶解度をその場測定した．この方法を用いれば，少量の材料で平衡化過程をその場観察できる．この手法をNH4Clの水に対する溶解度の圧力依存性の測定に適用し，他の手法による過去のデータと比較考察した．その結果，この手法は従来法に比べ，非常に少量のサンプルで測定でき，さらに精度の高い測定法であることが明らかになった．このことから，この手法はタンパク質のような，希少で高価な物質の溶解度を測定するのに適していると考えられる．


Interferometric study on the crystal growth of tetragonal lysozyme crystal

マイケルソン干渉計を用いて成長しているリゾチーム結晶の周りの濃度勾配を測定した．表面過飽和度(σsurf)を用いて結晶の成長モードを解析した結果，6<σsurf<20の間で二次元核成長モードであることが明らかになった．また，この広い過飽和度範囲において，リゾチームの拡散係数はほぼ一定で，4.9±0.7X10^(-11) m^2/sであることが分かった．


Observation of the concentration distribution around a growing lysozyme crystal

マイケルソン干渉計を用いて，成長しているリゾチーム結晶の周りの濃度分布をその場観察した．結晶の成長速度と濃度分布からリゾチームの拡散係数は1.59X10^(-10) m^2/sと見積もることができた．また粘性率を測定して，結晶に取り込まれる溶質のストークス半径を求めた結果，結晶に取り込まれる溶質の大きさはリゾチーム単分子と等しく，リゾチームの単分子が結晶の成長単位であることがわかった．


Highly Purified Glucose Isomerase Crystals Under Microgravity Conditions Grow as Fast as Those on the Ground Do

Suppression of convection flows (solute transportation) and that of impurity incorporation into crystals seem to be the main reasons why the quality of protein crystals becomes better under microgravity conditions, whereas each precise mechanism has not been completely clarified yet. We tried to clarify the former reason by the in-situ observation of spiral growth hillocks on the {110} faces of highly purified glucose isomerase (GI) crystals under microgravity conditions and on the ground. Lateral growth rates Vlateral of a spiral hillock on the {110} face of a glucose isomerase crystal in situ under microgravity conditions and step velocities Vstep of the same configuration on the ground took similar values as far as the maximum values are compared each other. This similarity indicates there are less influences of the convection flows on the growth rates of protein crystals contrary to conventional expectations.


Precipitant-free lysozyme crystals grown by centrifugal concentration reveal structural changes

The three-dimensional (3D) structure of a protein molecule in its crystal need not correspond to that found in vivo in many cases, since we usually crystallize protein molecules using precipitants (salts, organic solvents, polymeric electrolytes, etc.), and the precipitants are often incorporated into crystals along with the protein molecules. Although precipitant-free crystallization methods would solve these problems, such methods had not yet been established. We have achieved a novel precipitant-free crystallization method by liquid-liquid phase separation during the centrifugal concentration of lysozyme in ultra-pure water. In the 3D structure of the precipitant-free crystal, lysozyme loses a sodium cation and changes the position of Ser 72. Deionization of the solution also appears to induce a change in the position of Asp 101 and an increase in the activity of lysozyme.


Gravitational Tempering in Colloidal Epitaxy To Reduce Defects Further

Less-defective colloidal crystals can be used as photonic crystals. To this end, colloidal epitaxy was proposed in 1997 as a method to reduce the stacking defects in colloidal crystals. In this method, face-centered cubic (fcc) (001) stacking is forced by a template. In fcc (001) stacking, in contrast to fcc {111} stacking, the stacking sequence is unique, and thus the stacking fault can be avoided. Additionally, in 1997, an effect of gravity that reduces the stacking disorder in hard-sphere (HS) colloidal crystals was found. Recently, we have proposed a gravitational tempering method based on a result of Monte Carlo (MC) simulations using the HS model; after a colloidal crystal is grown in a relatively strong gravitational field, the defects can be reduced by decreasing the gravity strength and maintaining it for a period of time. Here, we demonstrate this method using MC simulations with a programmed gravitation. The dramatic disappearance of defect structures is observed. Gravitational tempering can complement gravitational annealing; some defect structures that accidentally remain after gravitational annealing (keeping the colloidal crystal under gravity of a considerable constant strength) can be erased.


Enlargement of Grains of Silica Colloidal Crystals by Centrifugation in an Inverted-Triangle Internal-Shaped Container

We successfully fabricated large grains of silica colloidal crystals in an inverted-triangle internal-shaped container (inverted-triangle container) by centrifugation. The largest grain in the container was much larger than that in a container which has a flat bottom and constant width (flat-bottomed container). The edged bottom of the inverted-triangle container eliminated the number of the grains, and then the broadened shape of the container effectively widened the grains.


Possibility of Gravitational Tempering in Colloidal Epitaxy to Obtain a Perfect Crystal

We have performed Monte Carlo simulations of hard spheres on a pattern under gravity. We have found that a crystal formed at a moderate gravity strength contains essentially no defects while one formed at a higher strength gravity contains a significant amount of defects. This result suggests the possibility of using gravitational-tempering in a colloidal epitaxy to reduce the number of defects in colloidal crystals. Moreover, we wish to emphasize the possibility to obtain a perfect crystal.


Significant Decrease in the Solubility of Glucose Isomerase Crystals under High Pressure

グルコースイソメラーゼ結晶の溶解度を0.1, 100 MPaでその場観察により測定した．具体的にはマイケルソン干渉計を用いてある濃度の溶液が結晶と平衡状態になるための平衡温度を，溶液濃度と圧力を変えて測定した．その結果，加圧に伴い溶解度が減少した．30℃で0.1から100 MPaまで加圧すると溶解度は1/9になった．このことはVisuriらによって報告された加圧による結晶化の促進を支持する．


Quick Fabrication of Gigantic Single-Crystalline Colloidal Crystals for Photonic Crystal Applications

センチメーターサイズのコロイド単結晶ドメインを，瞬間的なシアーフローを用いた濃厚懸濁液の非平衡配列のクエンチによって作製した．得られたコロイド結晶は，決まった結晶学的方位をもち，外界からの擾乱に対して十分に安定であった．この方法は，3次元フォトニック結晶の作製，応用に役立つであろう．

Japanese Journal of Applied Physics, Part 2 (Letters)


An interferometric study of the solubility of lysozyme crystals under high pressure

ニワトリ卵白リゾチーム正方晶系結晶に及ぼす圧力効果をマッハツェンダー干渉計と，我々のグループで新たにデザインした高圧光学セルを用いてその場観察により測定した．結晶の平衡化過程による濃度変化過程を，過飽和状態·未飽和状態の両方から正確にかつ連続的に測定した．溶解度の変化率はこれまでに測定されてきた値よりもずっと小さく，100 MPaで0.1 MPaの値の28%大きくなった．


Crystal Growth of Hen Egg White Lysozyme under High Pressure

ダイヤモンドアンビルセルを用いてニワトリ卵白リゾチームの結晶化に及ぼす静水圧の効果を研究した．結晶の核生成頻度と{110}, {101}面の成長速度を高圧力下でその場観察した．その結果，両方とも加圧に伴い減少した．観察された圧力効果はルシャトリエの原理で説明できる．


Interferometric Observation of the Interfacial Concentration Gradient Layers Around a Lysozyme Crystal

反射型マイケルソン干渉計を用いて，初めて，成長，もしくは溶解しているタンパク質結晶の表面モルフォロジー及び界面の濃度勾配層の観察に成功した．この方法を用いれば，溶液からの結晶の成長kineticsをうまく解析できるようになるであろう．