A procedure to analyze surface profiles of the protein molecules visualized by quick-freeze deep-etch replica electron microscopy

Quick-freeze deep-etch replica electron microscopy gives high contrast snapshots of individual protein molecules under physiological conditions in vitro or in situ. The images show delicate internal pattern, possibly reflecting the rotary-shadowed surface profile of the molecule. As a step to build the new system for the "Structural analysis of single molecules", we propose a procedure to quantitatively characterize the structural property of individual molecules; e.g. conformational type and precise view-angle of the molecules, if the crystallographic structure of the target molecule is available. This paper presents a framework to determine the observed face of the protein molecule by analyzing the surface profile of individual molecules visualized in freeze-replica specimens. A comprehensive set of rotary-shadowed views of the protein molecule was artificially generated from the available atomic coordinates using light-rendering software. Exploiting new mathematical morphology-based image filter, characteristic features were extracted from each image and stored as template. Similar features were extracted from the true replica image and the most likely projection angle and the conformation of the observed particle were determined by quantitative comparison with a set of archived images. The performance and the robustness of the procedure were examined with myosin head structure in defined configuration for actual application.     

Batch foam separation of a soluble protein

Removal of protein dissolved in water by batch foam separation was conducted with using ovalbumin (OA) as a model protein in the light of wastewater treatment reducing organic loading. The removal efficiency had a maximum value near the i.e.p. of OA (pH 4.6); thus, most experiments were conducted at pH 4.6. Typical experimental conditions; superficial gas velocity, U(g): 1.97 x 10(-2)-5.37 x 10(-2)cm/s; initial bulk concentration of OA, C(i): ca. 0.05-0.25 g/L; liquid volume, V: 600 cm(3). A model estimating bulk concentration profile was proposed by taking into account a mass balance of the present system. The model predicted that OA could be removed perfectly, however, was not all removed experimentally. The residual OA concentration of the bulk liquid within the column reached plateau value, which correspond to ca. 18% of the initial OA concentration. The plateau value of the bulk concentration was attained for ca. 100-500 min with U(g)=1.97 x 10(-2)-5.37 x 10(-2)cm/s. Foaming ability test revealed that the foaming limit concentration of OA at pH 4.6 was 9.72 x 10(-3)g/L. These results suggested that OA molecules could be damaged by interaction of bubble surface in the dispersed phase, since there were the residual OA concentrations over the limit concentration. To take account of this phenomena and correct the model, average surface density, X(d), which should convert protein molecule into the denatured protein molecule, was introduced. The corrected model could explain well the time profile of OA bulk concentration.     

Effect of pitting corrosion on strength of web plates subjected to patch loading

Pitting corrosion is typical corrosion observed on coated hold frames in way of cargo holds of bulk carriers, which exclusively carry coal and iron ore. Extensive survey on the effect of pitting corrosion on structural strength under a wide variety of loading conditions is necessary to clarify the relationship between pitting intensity and residual strength in detail. In the present study, a series of tests has been conducted on structural models which consist of web, shell and face plates to investigate the effect of pitting corrosion on strength of web plates subjected to patch loading. In these tests, artificial pitting was made on the web plates and two equal concentrated loads have been applied vertically at the one third points of simply supported models. It was found that web crippling behavior is strongly affected by the pit distribution on the web plates. According to the FE-analyses following the experiment, ultimate strength of the web plates with pitting under patch loading is a little smaller than or almost the same as that of the web plates with uniform corrosion in terms of average thickness loss.     

Accumulation of nitrous oxide in aerobic groundwaters

N₂O concentrations in the groundwaters collected in the Kanto District and Nagano Prefecture in Japan and five counties in New York State were determined. These N₂O data were obtained from the water samples from wells, springs and seepages from soils in forests and cropping fields. The N₂O concentrations in all samples greatly exceeded those of atmospheric equilibration. The average concentration of 690 nM N₂O is one order of magnitude larger than that in deep ocean. All groundwaters of the present study were aerobic with a high level of NO₃⁻, but with the absence of NO₂⁻ and NH₄⁺, and with a very low level of dissolved organic carbon. These characteristics suggest that the nitrate respiration in the aquifers is of little significance for the production of N₂O.

ΔN₂O/NO₃⁻ molar ratios in the groundwaters were between 10⁻⁴ and 10⁻² (Δ indicates the excess gas over that which would be in equilibrium). This supports the above view since the observed N₂O yield agrees with that reported for the production during an ammonia oxidation. If nitrification was indeed a major mechanism for the production of groundwater N₂O, subsequent release of N₂O from the aquifers that are polluted with nitrogen may deserve more close attention as a potential source of atmospheric N₂O via diffusion and discharge.     

Effect of substituting elements on hydrogen uptake for Pd–Rh–H and Pd–Ag–H systems evaluated by magnetic susceptibility measurement

The magnetic susceptibility and the pressure-composition isotherm were measured simultaneously for Pd–Rh–H and Pd–Ag–H systems in order to clarify the effect of Rh or Ag substitution on the hydrogen uptake from viewpoint of the electronic band structure. The magnetic susceptibility of all Pd binary alloys prepared decreased monotonically with increasing hydrogen content. At high hydrogen contents, the magnetic susceptibility became approximately zero for Pd–Rh–H and Pd–Ag–H system, and the hydrogen content at which the magnetic susceptibility gives zero corresponded with the terminal of the plateau region in the isotherm curve. The results indicated that the magnetic susceptibility of hydride phase was almost zero for all Pd binary alloys. On the basis of the band structure of Pd metal, we concluded that atom substitution only affected shift of the energy at Fermi level, and the amount of the hydrogen uptake was dominated by the number of unoccupied d-band in the alloys.     

Stabilized zirconia-based sensor utilizing SnO2-based sensing electrode with an integrated Cr2O3 catalyst layer for sensitive and selective detection of hydrogen

A highly selective hydrogen (H₂) sensor has been successfully developed by using an yttria-stabilized zirconia (YSZ)-based mixed-potential-type sensor utilizing SnO₂ (+30 wt.% YSZ) sensing electrode (SE) with an intermediate Al₂O₃ barrier layer which was coated with a catalyst layer of Cr₂O₃. The sensor utilizing SnO₂ (+30 wt.% YSZ)-SE was found to be capable of detecting H₂ and propene (C₃H₆) sensitively at 550 °C. In order to enhance the selectivity towards H₂, a selective C₃H₆ oxidation catalyst was employed to minimize unwanted responses caused by interfering gases. Among the examined metal oxides, Cr₂O₃ facilitated the selective oxidation of C₃H₆. However, the addition or lamination of Cr₂O₃ to SnO₂ (+30 wt.% YSZ)-SE was found to diminish the sensing responses to all examined gases. Therefore, an intermediate layer of Al₂O₃ was sandwiched between the SE layer and the catalyst layer to prevent the penetration of Cr₂O₃ particles into the SE layer. The sensor using SnO₂ (+30 wt.% YSZ)-SE coated with a catalyst layer of Cr₂O₃ as well as an intermediate layer of Al₂O₃ exhibited a sensitive response toward H₂, with only minor responses toward other examined gases at 550 °C under humid conditions (21 vol.% O₂ and 1.35 vol.% H₂O in N₂ balance). A linear relationship was observed between sensitivity and H₂ concentration in the range of 20–800 ppm on a logarithmic scale. The results of sensing performance evaluation and polarization curve measurements indicate that the sensing mechanism is based on the mixed-potential model.     

Investigation of UV-A light irradiation on tomato fruit injury during storage

We investigated the effect of UV-A light (wavelength 315 to 400 nm) irradiation during storage on tomato fruit injury. Mature green tomato fruit (cv. House Momotaro) were exposed to UV-A at doses of 0.02, 0.5, and 2 mW x cm(-2) throughout storage at 25 degrees C. The physiological disorders, fruit ripening, superoxide dismutase (SOD) activity, and increases in fruit temperature were evaluated. All UV-A-irradiated and nonirradiated tomatoes developed a full red color at the same time (2 weeks). Irradiated fruit ripened normally, and exposure of tomato fruits to UV-A did not lead to the discoloration of ripe tomato fruit at any dosage. The fruit temperature did not increase in response to various UV-A light doses and exposure times, and none of the UV-irradiated fruits showed physiological disorders (dull skin blemish, pitting). The SOD activity of UV-A-irradiated fruit exposed to the various UV-A doses did not significantly (P = 0.05) differ from that of fruit stored in dark conditions. The SOD results imply that UV-A light might not induce reactive oxygen species in UV-A-irradiated fruit.     

Purification and some properties of cholesterol oxidase stable in detergents from gamma-proteobacterium Y-134

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the gamma-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30 degrees C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3beta-ol (cholesterol) and 5alpha-cholestan-3beta-ol (dihydrocholesterol) at a high reaction rate, and the K(m) value for cholesterol was 65 microM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.     

Functional analyses of neutrophil-like differentiated cell lines under a hyperglycemic condition

Diabetic patients are prone to severe bacterial infections. The functional alterations of neutrophils by hyperglycemia are thought to be partially responsible for such infections. In this study, we investigated the functional changes of neutrophil-like differentiated cell lines (dHL-60, dTHP-1, and dNB-4) by treatment with 5.5 mM, 11 mM, or 35 mM of glucose. In dHL-60 cells, the incubation with high glucose (35 mM) resulted in the enhancement of cell aggregation, the suppression of cellular fragility, the induction of reactive-oxygen species (ROS) production by phorbol myristate acetate (PMA) stimulation, and the impairment of phagocytosis. In dTHP-1 cells, the treatment with higher glucose generated the suppression of cellular fragility and extremely impaired phagocytosis (by 35 mM), and induced ROS production due to PMA stimulation (by 11 mM). Furthermore, the higher glucose exposure to dNB-4 cells enlarged intracellular vacuoles (by 35 mM) and induced ROS production due to PMA stimulation (by 11 mM). Since the ROS generation of those cells was enhanced only after PMA stimulation under the higher glucose conditions, glucose may have a priming effect rather than a triggering effect. These extraordinary sensitivities caused by the higher glucose treatments may reflect the dysfunction or overactivation of neutrophils.     

水力机械模型试验台的设计研究

扼要介绍在水力机械模型试验台建设中,对试验台的主管路系统、流量率定系统、扭矩测量系统等进行的研究。开发了闸板可抽取式强迫压紧特殊阀,杜绝了管路内漏现象的发生;引进了模型水轮机叶片角度联调机构,在保证叶片角度调整精度的同时又提高了工作效率;采取了保持试验水质清洁的相关措施,节约了宝贵的水资源。该试验台通过了由中国电机工程学会和中国水力发电工程学会组织的技术鉴定,达到国际先进水平。