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The galectins are a family of β-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d -Glucose, d -allose, d -arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Modification of the domain architecture of galectins has been attempted to analyze their biological functions and to develop medical applications. Several types of galectin-1 repeat mutants were previously reported but, however, it was not clear whether the native structure of the wild type was retained. In this study, we determined the crystal structure of a galectin-1 tandem-repeat mutant with a short linker peptide, and compared the unfolding profiles of the wild type and mutant by chemical denaturation. The structure of the mutant was consistent with that of the dimer of the wild type, and both carbohydrate-binding sites were retained. The unfolding curve of the wild type with lactose suggested that the dimer dissociation and the tertiary structure unfolding was concomitant at micromolar protein concentrations. The midpoint denaturant concentration of the wild type was dependent on the protein concentration and lower than that of the mutant. Linking the two subunits significantly stabilized the tertiary structure. The mutant exhibited higher T-cell growth-inhibition activity and comparable hemagglutinating activity. Structural stabilization may prevent the oxidation of the internal cysteine residue.

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 β-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.

Galectin-9 is the most potent inducer of cell death in lymphomas and other malignant cell types among the members of the galectin family. We investigated the mechanism of galectin-9-induced cell death in PC-3 prostate cancer cells in comparison with in Jurkat T cells. Galectin-9 induced apoptotic cell death in Jurkat cells, as typically revealed by DNA ladder formation. On the other hand, DNA ladder formation and other features of apoptosis were not apparent in PC-3 cells undergoing galectin-9-induced death. Exogenous galectin-9 was endocytosed and destined to the lysosomal compartment in PC-3 cells. The internalized galectin-9 was resistant to detergent solubilization but was solubilized with lactose. Agents inhibiting actin filament dynamics abolished the internalization and cytocidal effect of galectin-9 in PC-3 but not Jurkat cells. Galectin-9 induced accumulation of ubiquitinated proteins, possibly heterogeneously ubiquitinated and/or monoubiquitinated proteins, in PC-3 cells. PYR-41, an inhibitor of the ubiquitin-activating E1 enzyme, suppressed the cytocidal effect of galectin-9. Although ubiquitination was upregulated also in Jurkat cells by galectin-9, PYR-41 was ineffective against galectin-9-induced cell death. Colocalization of ubiquitinated proteins and LAMP-1 was detectable in PC-3 cells treated with galectin-9. The ubiquitinated proteins were recovered in the insoluble fraction upon cell fractionation. In contrast, ubiquitinated proteins that accumulated after treatment with proteasome inhibitors did not co-localize with LAMP-1 and were mainly recovered in soluble fraction. The results suggest that atypical ubiquitination and accumulation of ubiquitinated proteins in lysosomes play a pivotal role in galectin-9-induced non-apoptotic death in PC-3 cells.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer-tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer-dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

We previously developed a stable form of galectin-9, an immunomodulatory animal lectin with a truncated linker peptide (G9Null), to overcome the protease sensitivity of wild-type galectin-9. G9Null is highly resistant to proteolysis, while the modification marginally improved the low solubility of the wild-type protein. To increase its solubility, we further modified the remaining linker region of G9Null. A 10-amino acid deletion with a single amino acid substitution resulted in an ∼400% increase in solubility and yield without an adverse effect on its biological activity. This mutant protein might be useful for large-scale recombinant production needed for evaluation of the therapeutic potential of galectin-9.

The STPR motif is composed of 23-amino acid repeats aligned contiguously. STPR was originally reported as the DNA-binding domain of the silkworm protein FMBP-1. ZNF821, the human protein that contains the STPR domain, is a zinc finger protein of unknown function. In this study, we prepared peptides of silkworm FMBP-1 STPR (sSTPR) and human ZNF821 STPR (hSTPR) and compared their DNA binding behaviors. This revealed that hSTPR, like sSTPR, is a double-stranded DNA-binding domain. Sequence-independent DNA binding affinities and α-helix-rich DNA-bound structures were comparable between the two STPRs, although the specific DNA sequence of hSTPR is still unclear. In addition, a subcellular expression experiment showed that the hSTPR domain is responsible for the nuclear localization of ZNF821. ZNF821 showed a much slower diffusion rate in the nucleus, suggesting the possibility of interaction with chromosomal DNA. STPR sequences are found in many proteins from vertebrates, insects, and nematodes. Some of the consensus amino acid residues would be responsible for DNA binding and concomitant increases in α-helix structure content.

観測方法の工夫や画像処理技術の進歩により，データオリエントなミクロ交通流モデルの構築が可能となってきている．筆者らの追従挙動モデルもその1つであり，実追従挙動が車間距離－相対速度平面上で描かれるスパイラル状曲線に従うことからバネ質点系の運動の類似としてモデル化されたものである．1 対の追従ペアに対する一定の再現性は既に確認されているが，その追従挙動モデル解の累積として生成される交通流が如何なる特徴を有するかの議論は課題とされた．交通流円滑化制御施策の検討等，工学的観点からもモデルパラメータと交通流現象との関係を知ることは極めて重要であり，本研究はこれを念頭に置きシミュレーション分析により本モデルパラメータの特性について考察することを目的とする．

Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.

高速道路における車両追従挙動を適切に表現できるモデルは未だ存在しないとされる．本研究では，実交通流中の走行試験から車間距離・相対速度平面上の軌跡がスパイラル状曲線となることに着目し，追従状態にある2台の車両のバネ質点系に希望車間距離に相当する強制外力が作用する非線形系の類似として捉えたモデルを記述する．さらにビデオ観測データを用いて検証を行なった結果，本モデルが車間距離・相対速度を良好に表現され，不特定多数のドライバーの追従挙動についても本モデルによって説明可能であることを示している．

Dislocation of the arytenoid cartilage occurs following medical instrumentation involving the laryngeal cavity or laryngeal injury from outside the larynx. We reported a case of spontaneously posterior dislocation of the arytenoid cartilage. A 53 year-old man suffering from suddenly recurring aphonia and its improvement many over 3 months without laryngeal injury or inducement eventually ceased to improve. Laryngoscopic findings showed that the left vocal fold was tensely prolonged and the vocal process of the arytenoid cartilage on the left side was dislocated posterolaterally. X-ray videofluorography of the larynx on repetitive phonation of /he/ showed abnormally high and diagonal displacement of the vocal fold and the upper structure of the arytenoid cartilage on the left side. Palpating the cricoarytenoid joint on the left side showed abnormal swelling with tenderness. Electomyography of the intrinsic laryngeal muscle on the left side showed normal action potential. From these findings, we diagnosed his voice disorder as spontaneously posterior dislocation of the arytenoid cartilage. We manually reduced it by pulling up a balloon inserted from the piriform sinus of the affected side to the esophagus.

A highly catalytic system for sugar oxidation in alkaline media is presented, for the first time, in which glucose oxidation takes place at ca. −0.44 V (vs. Ag|AgCl). Modification of Au(111) single crystal surface by under potential deposition (UPD) was carried out for a variety of metals and catalytic effect for sugar oxidation has been studied in 0.1 M NaOH. UPD of Ag ad-atoms on Au electrodes were of the best catalytic activity compared to other metals (Cu, Co, Ru, Cd, Ir, and Pt, etc.). For aldose type monosaccharide studied (glucose, mannose and xylose) as well as for aldose-containing disaccharides (maltose and lactose), one significant oxidation peak was obtained, however, no significant oxidation current was observed for disaccharides like sucrose. Gluconolactone and mannolactone gave no oxidation current at negative potentials at which glucose was oxidized, indicating no more than two-electron oxidation took place. With Ag ad-atoms coverage of ca. 0.3 monolayer leads to a positive catalytic effect expressed through a negative shift of ca. 0.14 V (glucose case) on the oxidation potential and a slight increase in peak current. At the Au(100) surface similar results to those at an Au(111) electrode were also observed. Keywords: Sugar oxidation, Electrocatalytic activity, Au single crystal electrodes, UPD, Fuel cells, Cyclic voltammetry

Galectin-9 is a lectin, which has various biological functions such as T-cell differentiation and apoptosis. Multivalency of carbohydrate binding is required for galectin-9 to function. Although galectin-1 (a proto-type galectin) forms an oligomer to obtain its multivalency, galectin-9 (a tandem-repeat-type one) has two carbohydrate recognition domains (CRD) in one polypeptide. However, a single CRD of galectin-9, especially the C-terminal one, exhibited pro-apoptotic activity suggesting oligomer formation capability. In this study, we monitored the nuclear magnetic resonance (NMR) signals of the backbone atoms of the galectin-9 C-terminal CRD (G9CCRD). Protein concentration dependence of the signals suggested that a region (F1-F4 strands) opposite to the ligand-binding site was involved in the self-association of G9CCRD. Site-directed mutagenesis in this region (Leu210, Trp277 and Leu279 to Thr; G9CCRD-3T) inhibited the self-association of G9CCRD, and improved the solubility, whereas it reduced its pro-apoptotic activity towards T cells. The high pro-apoptotic activity of G9CCRD seems to be due to the ability to form an oligomer. In addition, the same substitution in two-CRD-containing galectin-9 (G9Null-3T) also diminished the self-association and improved its solubility, although it hardly reduced the anti-proliferative and pro-apoptotic activities. G9CCRD contributes the self-association of full-length galectin-9 at high protein concentrations.

In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin.

We report the syntheses, structures, photophysical properties, and redox characteristics of the [2 + 2] pyromellitic diimide-based macrocycle with a linear pi-electronic system 2 as well as the 3,6-bis(phenylethynyl)pyromellitic diimide derivative 3. The interesting solid state structural properties of the clathrates of 3 with pi-donors are also reported. The macrocycle 2 was synthesized by the direct cyclocondensation followed by the Sonogashira coupling reaction. X-ray crystallographic studies showed that the phenylacetylene moieties in 2 formed the intramolecular benzene dimer structures, and the bis(phenylethynyl)pyromellitic diimide moieties in both 2 and 3 were stacked in a parallel and slanted arrangement. Theoretical calculations for 2' and 3 suggested the existence of electrostatic interactions between the bis(phenylethynyl)pyromellitic diimide moieties. The UV/vis spectral measurements and TDDFT calculations of 2, 2', and/or 3 were performed to understand their electronic transitions. The fluorescence spectral measurements showed that 2 and 3 have visible fluorescence properties and 2 displays an excimer fluorescence at ca. 590 nm. The cyclic voltammetry measurements revealed that the electrostatic repulsion between the diimide moieties in 2 is greater than that in 1 according to the extension of the pi-electronic systems. X-ray crystallography of the clathrates of 3 with various pi-donors demonstrated the formation of the segregated donor-acceptor structures, indicating the strong aggregation ability of the bis(phenylethynyl)pyromellitic diimide moiety.

Asparaginyl deamidation is a common form of nonenzymatic degradation of proteins and peptides. As it introduces a negative charge spontaneously and irreversibly, charge heterogeneity can be accumulated in protein solution during purification, preservation, and experiments. In this study, canine milk lysozyme (CML), a useful model for the study of the molten globule state, exhibited charge heterogeneity after sample purification. Four Asn residues in CML deamidated rapidly under mild conditions: pH 8.0 and 30 degrees C. Other than these residues, one Asn residue, which was stable in the native state, was labile to deamidation in the unfolded state. This suggests that the structural formation around Asn can suppress deamidation. Substitutions of these labile Asn residues to Gln residues prevented deamidation effectively. Because the substitutions did not disrupt the structural formation of the native and molten globule states, they will enable more precise analyses for physical and structural studies.

Due to the advancement of operative technic and radiation treatment and the development of ent has been noticed in the postoperative condition of total laryngectomy patients.Rehabilitation has zed people and the control of hygine also has been brought up as an important problem. In order to investigate the effect of elimi: ing nasal function which plays the role of humi ying and warming the incoming air, 18 tota laryngectomized people have been studied by m uring the surface temperature change in the tracl and broncheal mucosa.Many comparative stu have been carried out and many interesting gustions have been obtained.

Authos reported statistical findings of throat cancers among the Japanese in a period of past 11 years (from 1950 to 1960). The objective examination by enquete questionnaire to 46 university hospitals and 16 large hospitals, showd the total number of larynx cancers examined to be 2246 cases. The statistical observations were made from the following viewpoints, that is, age groups, sex distinction, yearly and local distribution, age and prognosis, interval from development to the first medical care, chief complains, clinical and histological classification, treatment, curative methods and prognosis, relationship between the prognosis and the types of cancer, recurrent and metastatic portions.

Lately in Japan, the registration system has been established.Therefore, statistical study of laryngeal cancer cases is now possible.In order to carry out the present study, many interviews of patients were made. Following are the main post-operative complications.1. permanent loss of speech function.2. naarowing of tracheostoma.3. dryness and crust formation in the region of tracheo-cutaneous junction and the upper tracheal area. 4. diminished of smell sense.5. abnormal taste sense.6. tendency to have frequent common colds.7. increased amount of sputum and occasional coughs.8. potential danger and inconvenience due to the location of newly created tracheal opening in the neck.9. shortness of breath.These post-operative complications beside No.1 were investigated on totally laryngectomized patients and the following reports were made.(1) Temperature on the mucosal surface of trachea and bronchi:Totally laryngectomized people must breath through the newly created opening in their neck. Generally speaking, the temperature on the mucosal surface is lower than normal and not stable.In addition, quick adjustment seems to be necessary to the sudden changes of enviromental temperature.(2) Histo-pathological study on tracheal and bronchial wall tissue.On autopsy, consecutive sections were made from tracheal and bronchial tissue of totally laryngectomized patients.Complete epithelial degener-ation, hyaline-like hyperplasia of basilar membrane and cell infiltration with fibrous proliferation took place in the upper portion of the trachea and carina where the changes of temperature were greater in degree.These histological changes correspond to the duration of tracheal respiration after surgery.(3) The usage of stoma cover on totally lar-yngectomized patients.In general, the tracheal opening incovered by 4 sheets of gauze (so-called apron) in order to protect the respiration. This stoma cover is effe-ctive in regulating surface temperature on tracheal and bronchial mucosa.In order to evaluate possible disturbances by using this stoma cover, pneumotachography was carried out which revealed no significant effects in function.(4) Smell sense:Smell test is carried out by intravenous inje-ction of Alinamin (B1 preparation), smell bottle and positive pressure nebulizer. All 7 cases show total loss of smell sense or highly reduced smell sense.(5) Taste sense :It seems to be difficult to summarize the con-dition of taste sense only by the conventional tests because taste disturbance is more complicated and delicate in nature. However, it is clear that 4 out of 5 show remarkable disturbance of taste sense.(6) Enlargement of tracheal opening:Narrowing of tracheal opening is not a common complication. Slight narrowing brings uncomforta-ble feeling but highly narrowed opening results in difficulty in breathing.In order to reduce this difficulty, partial removal of anterior portion of tracheal rings is performed.This operation shwos relatively good results.

C-type lysozyme catalyzes the hydrolysis of peptidoglycan of bacterial cell wall. Most of C-type lysozymes express to protect body against bacterial infection. However, ruminants and some leaf-eating animals have evolved their lysozyme as digestive enzyme. They recruit bacteria which ferment cellulose in the foregut, and digest the bacteria by lysozyme in the true stomach to obtain nutrient. Digestive lysozyme has acquired some properties, such as low optimal pH, and resistance to protease and acid hydrolysis. The structural basis for these properties still remains unclear. In this investigation, we have obtained the crystallographic structure of bovine stomach lysozyme (BSL). This is the first report on the structure of vertebrate digestive lysozyme. We have carried out the denaturant-unfolding experiment and revealed that BSL has high structural stability at acidic pH compared to non-digestive (hen egg-white) lysozyme. The structural stability in acidic solution would be related to the pepsin resistance which was previously reported for BSL. The crystal structure of BSL suggested that negatively charged surfaces, a shortened loop, and salt bridges should provide the structural stability.