Natural science is now extensively developed thanks to electron microscopy, but around 1950, only thin specimens were used because of the low accelerating voltages. The behavior of crystalline materials is very sensitive to specimen thickness, and thus in Japan a practical 500 kV electron microscope was constructed in 1965 for thicker specimens. It was shown that simultaneous reflection increases with increasing accelerating voltage, so that the maximum observable specimen thickness increases almost linearly up to 500 kV. Since simultaneous reflection becomes prominent above 1,500 kV, the in-situ observation of various phenomena representative of most bulk materials has been carried out with an ultra-high voltage electron microscope, whose accelerating voltages can reach 3,000 kV. Thus, even the characteristics of high-Z materials have been clarified in detail, and new applications, such as foreign atom implantation and the formation of non-equilibrium phases, have also been developed. The present account deals mainly with the importance of 3,000 kV electron microscopes, as applied to the new research fields of materials science.


(Contributed by Hiroshi FUJITA, M.J.A.)

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Since the stabilization of supercooled liquid in multi-component metallic alloys without any metalloid elements was discovered in 1988, a number of bulk glassy and nonequilibrium crystalline alloys have been fabricated with the aim of clarifying the origin of the novel stabilization phenomenon and searching for useful characteristics. This paper (Part 1) reviews our recent results on multi-component alloy systems in which the stabilization is achieved, some novel atomic configurations in stabilized supercooled liquids, the stabilization mechanism, and the physical, mechanical and chemical properties of the resulting bulk glassy alloys.


(Communicated by Yoshiaki ARATA, M.J.A.)

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The novel stabilization phenomenon of supercooled liquid in special multi-component metallic alloys that follow the three component rules has enabled us to fabricate a number of bulk glassy and nonequilibrium crystalline alloys exhibiting useful characteristics. Following the previous review (Part 1; Proc. Jpn. Acad. Ser. B 81, 156-171), this paper (Part 2) reviews our recent results on the physical, chemical, mechanical and magnetic properties of the resulting bulk nonequilibrium materials including glassy single phase alloys, nanocrystal-, nanoquasicrystal- and dendritic crystal-dispersed glassy alloys and nanocrystalline alloys. Finally, the application potential of bulk glassy alloys is addressed, taking account of their novel engineering properties and production processes.


(Communicated by Yoshiaki ARATA, M.J.A.)

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We have characterized novel glycosphingolipids (GSLs) of antigenic or functional importance, including type 3 and type 4 blood group ABH antigens, globo-series gangliosides, sialosyl dimeric Le^x, myeloglycan, β1-4GalNAc disialyl-Lc₄Cer, etc. Many GSLs have been identified as developmentally-regulated, tumor-associated antigens, suggesting their role in defining stage of development, and tumor cell phenotype. Out of the many types of GSLs, relatively few have been studied and shown to control cellular functions. Our studies indicate that functional effects of these GSLs are based on their interaction with specific target molecules in membrane, including (i) signal transducers (e.g., cSrc, Src family kinases, small G-proteins), to initiate signal transduction; (ii) integrin receptors (e.g., α3β1), to modulate cell adhesion and motility; (iii) growth factor receptors (e.g., for FGF, EGF), to modulate cell growth; (iv) tetraspanins (e.g., proteolipid CD9, CD81, CD82), to affect complex formation with integrin or with growth factor receptor; (v) GSL itself, through GSL-to-GSL interaction; (vi) microbial "adhesin". In many cases, these GSL interactions take place through GSL clusters at GSL-enriched microdomain (GEM). Some GEM show properties similar to those of "lipid rafts", whereas others, particularly those highly enriched in proteolipid/tetraspanin and involved in cell adhesion and cell growth, are distinguishable from "lipid rafts" since they are independent of cholesterol but are non-resistant to (soluble in) 1% Triton X-100. Such microdomains, showing GSL-dependent or -modulated cell adhesion and growth, are termed "glycosynapse". Further studies on GSL structure and function through glycosynapse will help clarify cell social behavior and various disease processes based on malfunction of cellular interaction, or of adhesion with concurrent signaling.


(Communicated by Tamio YAMAKAWA, M.J.A.)

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Thermoplasma volcanium is one of a small number of archaea able to live in anaerobic as well as aerobic environments. By sequencing spots in 2D gels, proteins expressed in this archaeon under the two conditions, aerobic or anaerobic, were identified. In the aerobic condition, types of proteins reducing active oxygens and the archaeal chaperonin refolding denatured proteins were identified as expressed in larger quantities. Two enzymes, L-asparaginase and an iron-molybdenium cluster-binding protein, were expressed, mediating pathways from nitrate to the TCA cycle, thereby activating aerobic proton transport for ATP synthesis. Under the anaerobic condition, a transcription factor in the FFRP family, TvFL5, and another protein making disulfide bonds in other proteins were expressed. So were types of enzymes that reduce anaerobic ferredoxin in order to activate anaerobic proton transport for ATP synthesis. Another type of ferredoxin containing zinc, and a flavoprotein, reducing possibly this ferredoxin, were expressed constitutively, probably for activating proton transport also in the aerobic condition. Five hours after changing the condition from aerobic to anaerobic, many of the aerobic proteins were still present, and in addition, the anaerobic proteins were expressed.


(Communicated by Masanori OTSUKA, M.J.A.)

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Monomeric sarcosine oxidase (MSOX) is a flavoprotein that catalyzes the oxidation of sarcosine to generate formaldehyde, glycine, and hydrogen peroxide, and is utilized in quantification of creatinine in serum. Crystal structure of MSOX from Bacillus sp. NS-129 (without ligand) has been determined at 1.86 Å Unlike the published structures of MSOX from Bacillus sp. B-0618 (without or with carboxylate-containing ligand), the two molecules in the asymmetric unit adopt distinct conformations at the active site loop (Gly56 to Glu60) with a maximal root-mean-square (RMS) displacement of 3.3 Å for C^α atom of Arg59. The multiple conformations seen at the active-site loop suggest that high flexibility of the loop would be important for the activity of MSOX.


(Communicated by Masanori OTSUKA, M.J.A.)

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Azoreductase AzoR is an oxidoreductase isolated from Escherichia coli as an enzyme responsible for the reduction of azo compounds. As the first step toward the elucidation of the molecular mechanism of function, we determined the three-dimensional structure of the enzyme by X-ray crystallography at 1.8 Å resolution. The crystal structure has revealed that AzoR is an FMN-containing and homodimeric enzyme. Each monomer consists of a twisted central parallel β-sheet surrounded on both sides by helices. The overall folding of the protein resembles NQO1, originally called DT-diaphorase [NAD(P)H: quinone reductase, EC 1.6.99.2], a mammalian FAD containing protein without significant sequence identity.


(Communicated by Masanori OTSUKA, M.J.A.)

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Flavin reductases (FRs) catalyze the reduction of free flavins using NADH or NADPH. In recent years, a new family of short-chain FRs have been identified in a variety of bacteria and archaea. Here we have determined the crystal structure of an archaeal short-chain FR, HpaC from an aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7. The HpaC molecule exists as a homodimer with one FMN for each monomer. On the other hand, PheA2, the most structurally similar protein to HpaC, contains FAD rather than FMN. Structural comparison of these proteins has revealed that the short loop at residues 82-83 and the successive η1 helix in HpaC are closer to the bound flavin than those in PheA2. As a result, there is not sufficient space to accommodate the AMP moiety of FAD in HpaC, and thus HpaC prefers FMN to FAD.


(Communicated by Masanori OTSUKA, M.J.A.)

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