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This paper presents two new clustering algorithms which are based on the entropy regularized fuzzy c-means and can treat data with some errors. First, the tolerance which means the permissible range of the error is introduced into optimization problems which relate with clustering, and the tolerance is formulated. Next, the problems are solved using Kuhn-Tucker conditions. Last, the algorithms are constructed based on the results of solving the problems.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5'-triphosphorylated, 2',5'-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2',5'-oligoadenylate synthetases (2',5'-OASs), inactivated by 5'-phosphatase and completely degraded by 2'-phosphodiesterase (2'-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2',5'-OASs and RNase L have been molecularly cloned and studied well, the identification of 2'-PDE has remained elusive. Here, we describe the first identification of 2'-PDE, the third key enzyme of the 2-5A system. We found a putative 2'-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2'-PDE by mass spectrometry. Based on the full-length sequence of the human 2'-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2'-PDE expressed in mammalian cells certainly cleaved the 2',5'-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2'-PDE were found, the human 2'-PDE was considered to be a unique enzyme without isoform. Suppression of 2'-PDE by a small interfering RNA and a 2'-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2'-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2'-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. We have previously obtained the mouse anti-Fas antibody HFE7A (m-HFE7A), which specifically induces apoptosis in inflammatory cells. In order to apply m-HFE7A for human therapy, we performed antibody humanization of m-HFE7A by grafting the mouse complementarity-determining regions (CDRs) to a human antibody. Five versions of humanized HFE7A (h-HFE7A) demonstrated the same antigen-binding affinity and same competition-binding activity against Fas as the chimeric HFE7A. Furthermore, these h-HFE7As induced the same degree of apoptosis in WR19L12a cells that express human Fas on their surface as chimeric HFE7A does. To further probe the structural basis for antibody humanization, we determined the three-dimensional structure of the h-HFE7A antigen-binding fragment (Fab) by X-ray crystallography and compared it with the crystal structure of the parent m-HFE7A Fab previously determined. The main-chain conformation in each h-HFE7A CDR is almost identical to that in m-HFE7A with root mean square (rms) deviations of 0.14-0.77 A. However, a significant segmental shift was observed in the CDR-L1 loop. Together with the high temperature factors of the CDR-L1 residues, both the loops are flexible, suggesting that the CDR-L1 loop would undergo conformational change upon binding to the antigen. Our results indicate that the humanization of m-HFE7A succeeded in maintaining the main-chain conformation as well as the flexibility of the CDR loop.

14C-Troglitazone was administered to male cynomolgus monkeys (monkey) orally or intraduodenally, and the time course of the plasma concentrations of the radioactivity and of the biliary excretions of the metabolites were investigated. The same was investigated for male marmosets after oral administration of 14C-troglitazone.1. The main metabolite in the plasma after oral administration to monkeys was the sulfoconjugate of troglitazone (M1). The quinone form metabolite (M3) and glucuronide (M2) of troglitazone were also detected in the plasma.2. The ratio of the biliary excretion of the radioactivity over a 24-hr period after intraduodenal administration to a bile-fistula cynomolgus monkey was 54.4%, which was as high as that observed in rats and dogs. The main metabolites in the bile were M1 and M2, each accounting for about 40% of the biliary radioactivity. An unknown metabolite, U3, was also detected at a ratio of about 6%. U3 was also detected in the bile after administration of M3, and was show to contain the quinone-like structure.3. The plasma metabolites after oral administration to marmosets were almost the same as those in the plasma of monkeys. U3 was also observed in the plasma of marmosets.4. U3 was isolated from the bile and urine of monkey and marmoset after administration of 14C-M3 or M3. According to the MS and NMR spectra, the chemical structure of U3 was determined as the glucuronide of the hydroquinone form of troglitazone, and its was conjugated at position 8"a.

The structure of a new anthocyanin from Salvia uliginosa petals has been identified as delphinidin 3-O-[6-O-(p-coumaroyl)-β-d-glucopyranoside]-5-O-[4-O-acetyl-6-O-malonyl-β-d-glucopyranoside] using mass spectroscopy and NMR. This compound constitutes the anthocyanin component of the pigment responsible for blue flower colour in S. uliginosa.

1-Deoxynojirimycin-trehalamine-fused and -linked compounds (10, 19a and 19b) were synthesized from 1-deoxy-2,3,4,6-tetra-O-benzylnojirimycin and trehazolamine, which was obtained from natural trehazolin as a degradation product. None of these synthetic compounds exceeded 1-deoxynojirimycin in the inhibitory activities towards rat intestinal maltase and yeast α- d -glucosidase.

Novel endothelin antagonists, nahocols A, A1, B, C, D1 and D2, and isonahocols D1 and D2, were isolated from the brown alga, Sargassum autumnale. Their structures were determined through detailed analysis of NMR spectra and chemical reactions. Nahocols have an aryl prenyl ether structure, which are speculated to be biogenetic precursors of the ubiquitous prenyl hydroquinones or prenyl benzoquinones in the plant and animal kingdoms.

The proposed structure of natural flavolipin was revised as N-[N-[(3R)-15-methyl-3-(13-methyltetradecanoyloxy)hexadecanoyl]glycyl]-L-serine as a result of synthetic study and biological activity test.

1-Deoxynojirimycin-trehalamine fused compound 10 as a mixture together with 11 and its related compound 19 were synthesized The enzyme inhibitory activities of the mixture ( 10 and 11 ), 19 and 20 exhibited IC 50 values of 0.68, 4.2, and 1.5 μg/ml, respectively, toward rat intestinal maltase.

A novel endothelin-converting enzyme (ECE) inhibitor, B-90063, was isolated from the culture supernatant of the newly discovered marine bacterium Blastobacter sp. SANK 71894. Based on spectral analyses and chemical reactions, the structure of B-90063 was determined to be bis[6-formyl-4-hydroxy-2-(2'-n-pentyloxazol-4'-yl)-4-pyridon -3-yl]-disulfide (1a). Human and rat ECEs were inhibited more potently by B-90063, with respective IC50 values of 1.0 and 3.2 microM, than were other neutral endopeptidases such as NEP and type-I and -IV collagenases. B-90063 also inhibited the binding of ET-1 to rat ET(A) and bovine ET(B) receptors, though its antagonistic activities were weak. B-90063, thus, may abolish the physiological actions of endothelins through the ECE inhibitory and receptor antagonistic mechanisms.

Tetrahydropyrano[2,3- d ]oxazole 6 as an analogue of trehazolin was synthesized from compound 3 , which is a protected 1,3-bis(β- d -glucopyranosyl)thiourea. In contrast, compound 10 , a protected 1,3-bis(α- d -glucopyranosyl)thiourea, and 15 , a mixed species having one α- d -glucopyranosyl group and one β- d -glucopyranosyl group, did not yield the corresponding pyranooxazolines as anticipated, giving instead, respectively, a furo[2,3- d ]oxazole 14 and a complex mixture of unidentified products.

Schizostatin (1) has been isolated as a potent and selective inhibitor of squalene synthase. Its structure has been determined using spectroscopic methods: the compound is shown to be a diterpenoid which has a trans-dicarboxylic acid moiety. Total synthesis of Schizostatin (1) was achieved by the highly regio- and stereo selective coupling reaction of an allylic bromide with a barium reagent. The Z-isomer 16 was also prepared using the stereo selective syn-addition of an organocopper reagent to acetylenedicarboxylate.

A novel acyl-CoA : cholesterol acyltransferase (ACAT) inhibitor, designated epi-cochlioquinone A has been isolated from the fermentation broth of Stachybotrys bisbyi SANK 17777. The molecular formula, physicochemical properties, NMR spectroscopic analysis and X-ray crystallographic analysis revealed that this compound was a stereoisomer of cochlioquinone A, which has been previously reported as a nematocidal agent. It inhibited ACAT activity in an enzyme assay using rat liver microsomes with an IC50 value of 1.7 microM. However, it showed about 10-fold less potent inhibitory effect on plasma lecithin cholesterol acyltransferase (LCAT) than on ACAT. In addition, it inhibited in vivo cholesterol absorption in rats by 50% at 75 mg/kg.

The absolute configuration of Leustroducsins was determined to be 4 S , 5 S , 8 R , 9 R , 11 R , 16 R , 18 S by the modified Mosher's method.

6-epi-Trehazolin was synthetized in a stereocontrolled manner, and this synthesis proved that an oxazine structural isomer of 6-epi-trehazolin does not exist

Diterpenes phomactins B (1a), B1 (1c), B2 (2a), C (4), and D (5) were isolated from a marine fungus, Phoma sp. These structures were determined based on spectroscopic evidences, X-ray crystallography, and chemical conversion. We also determined the absolute configuration of 1a, 1c, and 2a by the advanced Mosher's method and circular dichroism. Phomactin D inhibited the binding of PAF to its receptors and PAF-induced platelet aggregation with IC 50 of 1.2×10 -7 M and 8.0×10 -7 M, respectively, while other compounds antagonized PAF action at higher concentrations

6-Epi-trehazolin was synthesized in a stereocontrolled manner, and this synthesis provided proof for the non-existence of oxazine structural isomer of 6-epi-trehazolin.

The three-dimensional structure of leualacin, a novel calcium channel blocker from Hapsidospora irregularis was determined in CDCI 3 . Based upon the dihedral angle constraints from the analysis of 3 J H,H and 3 J C,H , and the distance constraints deduced from 1 H-{ 1 H} NOEs and 13 C- { 1 H} NOES, conformers completely satisfying the NMR data were obtained by the conformational grid search program SYBYL followed by the energy minimization program XPLOR. Leualacin's structure is characterized by γ- and β-turn like moieties analogous to cyclic peptides, which are fixed by trans-annular hydrogen bonds formed between L-leucine and β-alanine. L-N-methylphenylalanine and S-leucic acid were found to be connected by cis peptide bond. The RMSDs (Root Mean Square Differences) calculated for the backbone atoms among four structures are 0.6 A. The ring current effect caused by the phenylalanine moiety was reproduced by a calculation based on the resulting structures according to Bovey and Johnson's formula.

3-Thiazoliomethylcephalosporin 1 was transformed under alkaline conditions into 3-spirocephalosporin 6. The structures of three isolated stereoisomers were elucidated by NMR experiments.

The structure of a novel herbicide, hydantocidin isolated from the fermentation broth of Streptomyces hygroscopics SANK 63584, was determined by the combined analysis of MS and 1H NMR spectra. Hydantocidin is a novel spiro compound containing a subofuranoid ring, at the anomeric position of which a hydantoin ring is used such that the C(1)–N(1) linkage is β. The relative configuration and the conformation in solution was determined by quantitative analysis of the NOE spectra and T1 values. In CD3OD and [2H6]DMSO (dimethyl sulphoxide) solutions, the ribofuranose moiety of hydantocidin was found to be fixed in a C2-endo conformation, probably due to the rigidity of the spiro structure and hydrogen bonding between 3-OH and the carbonyl group at C-4′.

Diterpenes phomactins B (1a), B1 (1c), B2 (2a), C (4), and D (5) were isolated from a marine fungus, Phoma sp. These structures were determined based on spectroscopic evidences, X-ray crystallography, and chemical conversion. We also determined the absolute configuration of 1a, 1c, and 2a by the advanced Mosher's method and circular dichroism. Phomactin D inhibited the binding of PAF to its receptors and PAF-induced platelet aggregation with IC 50 of 1.2×10 -7 M and 8.0×10 -7 M, respectively, while other compounds antagonized PAF action at higher concentrations

6-epi-Trehazolin was synthetized in a stereocontrolled manner, and this synthesis proved that an oxazine structural isomer of 6-epi-trehazolin does not exist

Synthesis of 5-epi-trehazolin (trehalostatin) (2) was accomplished via the crucial intermediate, epoxide (6 alpha), from D-glucose. The stereochemistry of epoxide (6 alpha) and its isomer (6 beta) which were obtained from Sharpless epoxidation, was determined by comparison between the NMR relaxation times of relevant protons.

The novel schizostatin ( 1 ) has been isolated as a potent inhibitor of squalene synthase. Its structure elucidation and total synthesis are described. Synthesis of the Z-isomer 12 and its biological activity are also reported.

The absolute configuration of Leustroducsins was determined to be 4 S , 5 S , 8 R , 9 R , 11 R , 16 R , 18 S by the modified Mosher's method.

Tetrahydropyrano[2,3- d ]oxazole 6 as an analogue of trehazolin was synthesized from compound 3 , which is a protected 1,3-bis(β- d -glucopyranosyl)thiourea. In contrast, compound 10 , a protected 1,3-bis(α- d -glucopyranosyl)thiourea, and 15 , a mixed species having one α- d -glucopyranosyl group and one β- d -glucopyranosyl group, did not yield the corresponding pyranooxazolines as anticipated, giving instead, respectively, a furo[2,3- d ]oxazole 14 and a complex mixture of unidentified products.

A novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor, designated epi-cochlioquinone A has been isolated from the fermentation broth of Stachybotrys bisbyi SANK 17777. The molecular formula, physicochemical properties, NMR spectroscopic analysis and X-ray crystallographic analysis revealed that this compound was a stereoisomer of cochlioquinone A, which has been previously reported as a nematocidal agent. It inhibited ACAT activity in an enzyme assay using rat liver microsomes with an IC50 value of 1.7μM. However, it showed about 10-fold less potent inhibitory effect on plasma lecithin cholesterol acyltransferase (LCAT) than on ACAT. In addition, it inhibited in vivo cholesterol absorption in rats by 50% at 75mg/kg.

1-Deoxynojirimycin-trehalamine fused compound 10 as a mixture together with 11 and its related compound 19 were synthesized The enzyme inhibitory activities of the mixture ( 10 and 11 ), 19 and 20 exhibited IC 50 values of 0.68, 4.2, and 1.5 μg/ml, respectively, toward rat intestinal maltase.

The proposed structure of natural flavolipin was revised as N-[N-[(3R)-15-methyl-3-(13-methyltetradecanoyloxy)hexadecanoyl]glycyl]-L-serine as a result of synthetic study and biological activity test.

A-53930A, B and C, which inhibit N-type Ca2+ channels, were isolated from the culture broth of Streptomyces vinaceusdrappus SANK 62394. A-53930A and B were new compounds which contained a carbamoyl group on the 6-hydroxyl group of the D-gulosamine part of streptothricin. A-53930C was identical to streptothricin B. A-53930A, B and C inhibited [125I]omega-conotoxin MVIIA binding to N-type Ca2+ channels (IC50= 0.17, 0.091 and 0.071 microM), but did not inhibit [3H]PN200-110 binding to L-type Ca2+ channels (IC50 > 50 microM). These compounds also inhibited [3H]norepinephrine release from chick cerebral cortex synaptosomes (IC50=91.0, 20.6 and 39.5 microM), indicating these compounds selectively block N-type Ca2+ channels which are important for neurotransmitter release. It was also revealed that although A-53930C had antimicrobial activity against gram-negative and -positive bacteria and fungi, A-53930A and B showed weak activity only against gram-negative bacteria.

The proposed structure of natural flavolipin was revised as N-[N-[(3R)-15-methyl-3- (13-methyltetradecanoyloxy)hexadecanoyl]glycyl]-L-serine as a result of a synthetic study and biological activity tests, and its isomers were synthesized in a stereocontrolled manner.

1-Deoxynojirimycin-trehalamine-fused and -linked compounds (10, 19a and 19b) were synthesized from 1-deoxy-2,3,4,6-tetra-O-benzylnojirimycin and trehazolamine, which was obtained from natural trehazolin as a degradation product. None of these synthetic compounds exceeded 1-deoxynojirimycin in the inhibitory activities towards rat intestinal maltase and yeast α- d -glucosidase.

Biosynthesis of griseolic acids, competitive inhibitors of cyclic nucleotide phosphodiesterase, was investigated with the culture of a producing strain of Streptomyces griseoaurantiacus. 13C-Labeled and 15N-labeled compounds were added into the culture, and 13C-enriched and 15N-enriched griseolic acid A was isolated from the culture medium and analyzed by 13C NMR and 15N NMR spectroscopy. The compounds added to growth medium were [2-13C]acetate, [1, 2-13C] acetate, [1, 4-13C]succinate, [1-13C]glucose, [6-13C]glucose, [2-13C]ribose, and [1-13C, 15N]glycine. The results suggest that adenosine, which is formed from amino acids and sugars contributes the adenine and ribose moieties to griseolic acid A. The data also suggest that a dicarboxylic acid from the Krebs tricarboxylic acid cycle contributes to the dicarboxylic part of the compound.

A strain of actinomycetes identified as Saccharothrix sp. SANK 64289 was found to produce new antibiotics, galacardins A and B. Their physico-chemical properties showed that they were new members of glycopeptide antibiotics. They were structurally related to beta-avoparcin but differed from it only in sugar composition. Though beta-avoparcin does not contain galactose, galacardins A and B did contain two and one moles of galactose, respectively. They showed strong antimicrobial activity against Gram-positive bacteria and also showed excellent in vivo protective activity against Staphylococcus aureus infection in mice.

A new inhibitor of phospholipase A2 was isolated from the fermentation broth of Aspergillus unguis. The structure, with a depsidone carbon skeleton, was assigned by spectroscopic experiments.

The spiro-hydantoin ring at the anomeric position of D -and L -furanose was constructed by using aldol addition followed by acid promoted cyclization and the synthesis of thestereoisomeis of (+)-hydantocidin, L - and D -series of spiro-furanose 2, 3, 4 and 5.

This paper presents two new clustering algorithms which are based on the entropy regularized fuzzy c-means and can treat data with some errors. First, the tolerance which means the permissible range of the error is introduced into optimization problems which relate with clustering, and the tolerance is formulated. Next, the problems are solved using Kuhn-Tucker conditions. Last, the algorithms are constructed based on the results of solving the problems.

Kernel functions are remarked in the field of clustering or pattern recognition. Using the functions, good results of clustering can be obtained for data with hard nonlinear distribution. But there is the problem that the calculation cost increases using the kernel functions. In this paper, new hierarchical clustering algorithms using kernel functions are proposed. Moreover, the availability of proposed algorithms is discussed through some numerical examples. The proposed methods do not include the kernel functions positively so that the methods can be introduced into the existing resources easily.

A humanized version of the apoptosis-inducing mouse anti-human Fas monoclonal antibody, HFE7A, is under further development for the treatment of autoimmune diseases such as rheumatoid arthritis. We have crystallized the antigen-binding fragment (Fab) of the humanized HFE7A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 54.4 A, b = 82.7 A, c = 104.9 A and contain one Fab molecule in the asymmetric unit. X-ray diffraction data were collected to 2.8 A resolution.

The Fas-Fas ligand system is involved in apoptosis. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A) has a potential use in human therapy against autoimmune diseases such as rheumatoid arthritis. Information on the three-dimensional structure is essential for antibody humanization. Crystals of an antigen-binding fragment (Fab) of m-HFE7A were obtained by the hanging-drop vapour-diffusion method using sodium citrate as a precipitant and 2-methyl-2,4-pentanediol as an additive. Fast optimization to produce single crystals suitable for X-ray analysis was achieved by the streak-seeding technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.4, b = 74.0, c = 133.8 A. The crystals diffract at least to 2.5 A resolution.

A-53930A, B and C, which inhibit N-type Ca2+ channels, were isolated from the culture broth of Streptomyces vinaceusdrappus SANK 62394. A-53930A and B were new compounds which contained a carbamoyl group on the 6-hydroxyl group of the D-gulosamine part of streptothricin. A-53930C was identical to streptothricin B. A-53930A, B and C inhibited [125I]omega-conotoxin MVIIA binding to N-type Ca2+ channels (IC50= 0.17, 0.091 and 0.071 microM), but did not inhibit [3H]PN200-110 binding to L-type Ca2+ channels (IC50 > 50 microM). These compounds also inhibited [3H]norepinephrine release from chick cerebral cortex synaptosomes (IC50=91.0, 20.6 and 39.5 microM), indicating these compounds selectively block N-type Ca2+ channels which are important for neurotransmitter release. It was also revealed that although A-53930C had antimicrobial activity against gram-negative and -positive bacteria and fungi, A-53930A and B showed weak activity only against gram-negative bacteria.

Cytokine-induced neutrophil chemoattractant (CINC/Gro) is a chemotactic factor for neutrophils in the rat and a member of the CXC chemokine family. The refined three-dimensional structure of CINC/Gro was derived with the aid of heteronuclear magnetic resonance spectroscopy and a hybrid method of distance geometry and simulated annealing. The backbone atomic r.m.s. differences for 19 structures about the mean coordinates for residues 7 to 70 are 0.69+/-0.15 A for the dimer and 0.56+/-0.13 A for the monomer. The N terminal region containing an ELR sequence, an essential motif for chemotactic activity, is disordered in solution, as in other CXC chemokines. The dimer structure consists of a six-stranded anti-parallel beta sheet with two C-terminal alpha helices on the beta sheet. The overall dimer structure of CINC/Gro is similar to that of human MGSA, though the backbone r.m.s.d.s between CINC/Gro and the two MGSA structures were high (1.81 and 2.34 A for the monomer) in spite of the high sequence homology (67%). The major difference resides in the relative position of the C-terminal alpha helix with respect to the beta sheet. This results in a difference of interhelical distances in the dimer: wider (15 A) in CINC/Gro, as in IL-8, than in MGSA (11.7 and 10 A).