Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

Decomposition of polyurethane foams by alkanolamines

Methylene diphenyl isocyanate (MDI)-based polyurethane flexible foams were decomposed by alkanolamines without a catalyst at 150°C. The decomposition products were completely separated into two layers. The upper liquid layer was a polyether polyol, and the lower liquid layer was methylene diphenyl amine (MDA) and alkanolamine derivatives. These layers were analyzed using gel permeation chromatography (GPC) and ¹³C-NMR. The upper layer was obtained as a relatively pure polyether. The lower layer was observed to be MDA and 2-hydroxyethyl carbamic acid ester prepared from alcoholysis products during the decomposition reaction. It was thought that the decomposition process of polyurethane foam by alkanolamines was not aminolysis but alcoholysis. © 1994 John Wiley & Sons, Inc.     

Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).     

The conversion of a field-emission scanning electron microscope to a high-resolution,high-performance scanning transmission electron microscope,while maintaining original functions

Recently, the reliability of field-emission electron guns has increased. In addition, the cost of computer systems for on-line processing has dropped. Hence, we should now consider the use of scanning transmission electron microscopy (STEM) for routine work, especially, in the field of biology where one may expect to utilize digital image processing techniques. An STEM has been constructed, without disturbing the original functions, by converting a commercial scanning electron microscope equipped with a fieldemission gun. The STEM is generally operated at accelerating voltage 30 kV, focal length 7.5 mm, and beam current 1?2 × 10^?10 A. Several improvements have been incorporated for removing the effects of vibration, contamination, and stray magnetic fields. Also, an adjustable detector aperture was utilized. The modified instrument was connected to an on-line digital image processing system for utilizing the information obtained from STEM images. The advantages of the modified system were studied from various viewpoints.     

Simultaneous SAXS/WAXS experiments on shear induced iPP crystallization near nominal melting temperature

Simultaneous small- and wide-angle synchrotron radiation X-ray scattering was performed to monitor the evolution of crystalline structure within the iPP melt during and after applying pulse shear. The iPP melt was subjected to a strong pulse shear of 240 s^?1 for 6 s at temperatures between 150 and 170 °C below and above the nominal melting temperature T_m. It was found that the imposed shear affected the melt only at lowest temperature. Structures generated during flow above 150 °C were too dilute to be detected. To extract the hidden structure, the melt was cooled to crystallization temperature of 150 °C either immediately after shearing or after annealing at shear temperature for up to 30 min. This treatment manifested with an anisotropic structure in a few minutes after quenching, undetectable when both shear and annealing temperatures presented the same value. The data obtained revealed also close correlations between annealing time, shear temperature and incubation time.     

Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon

We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30 degreesC and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E. coli RNase HI. The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6. 8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HIIPk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.     

Surface adsorption of propane, propylene and propane/propylene mixed gas on polyimide observed with X-ray and neutron reflectivity

We observed the surface adsorption layer of propylene, propane and propane/propylene mixed gas on a polyimide film as a function of pressure using X-ray (XR) and neutron (NR) reflectivity. It was initially confirmed by NR that the surface layer is an adsorption layer of propylene using deuterated gas. We have determined by XR the adsorption isotherms of these gases and found that the thickness of the surface adsorption layer of propane and propane/propylene mixed gas is much larger than that of propylene and the isotherm of propane/propylene mixed gas is similar to that of propane. The former and the latter findings predict that the affinity of propane to the polyimide is greater than propylene and the content of propane is much higher than that of propylene in the adsorption layer. We also evaluated the permeability coefficients of these gases as a function of the applied pressure, and found that the permeability coefficients begin to increase at a pressure corresponding to the onset of the adsorption layer. This suggests that the surface adsorption layer of the gas plays an important role in the hydrocarbon gas permeation mechanism in a polymer membrane, while a swelling of the polyimide film is the basis for the increase in the permeability with pressure.     

Relationship between the local dynamics and gas permeability of polyacetylenes containing polymethylated indan/tetrahydronaphtalene moieties

Local dynamics of polyacetylenes having polymethylated indan/tetrahydronaphthalene moieties have been studied using quasielastic neutron scattering, and the results obtained have been discussed together with former results with various poly(diphenylacetylene) derivatives. Clear quasielastic scattering was observed for the present polymers although they were in the glassy state, and the observed dynamic scattering laws were well fitted to a model function assuming the sum of narrow and broad Lorentzians. Both the relaxation rates and fractions from the narrow component exhibited a positive correlation with oxygen permeability coefficients (PO₂)$\left(P_{{\text{O}}_2}\right)$, while those from the broad component hardly related to PO₂$P_{{\text{O}}_2}$. Furthermore, the local flux, which is defined as the product of fraction and relaxation rate of the narrow component, is also found to be proportional to PO₂$P_{{\text{O}}_2}$. These findings imply that only the narrow component contributed to the gas permeability. Thus, it has been clarified that the local dynamics with a time scale of several tens of picoseconds plays a role of key importance for the gas permeability of substituted polyacetylenes in the glassy state.     

Annealing effects on thickness of polystyrene thin films as studied by neutron reflectivity

We performed neutron reflectivity measurements on deuterated polystyrene thin films supported on silicon substrate as a function of temperature. In order to see effects of annealing on the thickness, the films were annealed at 80 °C for 12 h and 135 °C for 12 h, termed weakly and strongly annealed films, respectively. One of the main purpose of this study is to see if the negative expansivity reported for very thin films [Phys. Rev. Lett. 71 (1993) 867] is caused by unrelaxed structure due to lack of annealing. It was found that the weakly annealed films show negative expansivity in the glassy state and it disappears for the strongly annealed films with thickness above about 90 Å. This suggests that the negative expansivity is due to the unrelaxed structure. In addition to this relaxation process, the thickness difference between the heating process and the cooling process suggests that there is another very slow relaxation process in thin films detectable at around 135 °C or about 32 °C above the glass transition temperature T_g. As a candidate for this slow process a sliding motion proposed by de Gennes is discussed.