Paecilomyces tenuipes, which was also called Isaria japonica, is one of the famous entomopathogenic fungi. This fungus is used as a treatment of various diseases and an activator of physiological functions in Chinese from ancient times. It is very rare and expensive because of difficulty of its collection in the nature. We cultivated fruit body of Paecilomyces tenuipes on a large scale, and isolated four novel highly oxygenated trichothecanes 1-4 from the methanol extract of it.. 1 and 2 are the first example of trichothecanes which possess a spiro ring. 1-3 showed moderate neuronal differentiation activity on PC-12 cells. It is interesting that the production of trichothecanes was dependent on culture conditions. Some trichothecanes are famous as mycotoxins, therefore, the isolation of this type of compounds suggested that artificially cultivated fruit body of Paecilomyces tenuipes should be carefully used as medicines and medicinal health food.

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We have achieved a novel effective approach to chiral cyclobutanones(3) via Sharpless asymmetric dihydroxylation of cyclopropylidene derivatives followed by 1,2-rearrangement. The results show that the disubstituted derivatives are better substrates than the monosubstituted ones. Of these disubstituted cyclopropylidene derivatives, (1e) having bulky substituent on ortho position of aromatic ring, showed moderate enantioselectivity to give the optically active cyclobutanone (3e) in high yield, which constitutes a total formal synthesis of (-)-filiformin. Furthermore, we examined a synthesis of trichothecane antibiotics along with this cyclobutane strategy. A synthesis of A-ring aromatic trichothecane (13) was achieved via palladium mediated ring expansion of (9) and regiocontrolled cyclization of (12) as key steps. The compound (13) thus preprared was controlled into functionalized diene (16) via diol (14) and acetonide (15). The studies for the conversion of (16) into scirpene are now under progress.

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Three novel trichothecene sesquiterpenes, trichothecinols A (1), B (2) and C (3) were isolated from the fungus Trichothecium roseum (TMI-32358) together with some known analogues such as trichothecin (4). They showed inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and thus were classified as anti-tumor promoters in vitro. We focused on the potent activity of 1 and further examined its inhibitory effect on the tumor promotion in mouse skin in vivo. Surpprisingly, while 1 reduced both the rate of papilloma-bearing mice and the number of papillomas per mouse in the presence of TPA, 1 itself enhanced the tumor promotion in the absence of TPA and induced the formation of papillomas. In addition, malignant conversion of papillomas to carcinomas was caused after 20 weeks without 1. On the other hand, 1 did not enhance the tumor promotion in vitro in the absence of TPA at all. Accordingly, 1 was indicated to be a new class of tumor promoter that can be distinguished from TPA and teleocidin. In order to clarify the peculiar action of 1 in the tumor promotion, a structure-activity relationship was investigated with the natural products and compounds derived from 1 and 4. In anti-tumor promoting effect in vitro, it was found that the presence of the isocrotonyl ester and conjugated ketone was most important for increasing the activity. The presence of the 3-hydroxyl group was also effective when the ketone carbonyl group was reduced. Interestingly 4 did not enhance the tumor promotion neither in vitro nor in vivo at all although the structure of 4 differs from 1 only in the lack of 3-hydroxyl group and its antitumor promoting activity in vitro was comparable to that of 1. Therefore, it was strongly suggested that the 3-hydroxyl group plays a crutial role in the tumor promotion.

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Pesticides included in cyclodextrin (CD) possess the following excellent properties: 1. stabilization of labile compounds and improvement of residual activity, 2. applicability in powdery formulations, 3. selectivity between target pests and beneficial species by introducing the stomach poisonrelated biological properties additionally, 4. reduction of phytotoxicity, 5. reduction of dermal toxicity.As shown in the cases of Pyrethrins CD 5 % WP and Pyrethrins CDN Dust containing 0.1% pyrethrins plus 2% carbaryl, they were proved to be useful in practical applications. At present, the cyclodextrins are on the market mainly for use in fine chemical products such as pharmaceuticals and foods. The application of the inclusion techniques to the bulky agricultural fields requires the development of CD manufacturing processes for large-scale production leading to considerable cost reductions. It is hoped therefore that studies in this area will be encouraged.

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Rice a-amylase isozymes, AmylA and Amy3D, were expressed by Saccharomyces cerevisiae. Reaction properties of those isozymes purified by immuno-affinity chromatography were characterized to elucidate their functional roles. The pH optima of AmylA (pH 4 .2) and Amy3D (pH 5.5) correlate with the pH value of the endosperm tissue at times in rice seedling development when these isozymes are expressed. AmylA showed higher reactivities to soluble starch and starch granules than Amy3D. On the other hand, Amy3D showed much higher reactivity to maltoheptaose than AmylA. These results suggest that the individual isozymes play different functional roles during the seedling development process. A mutant enzyme, [N240Q]AmylA, which does not have an N-linked carbohydrate chain was created by site-directed mutagenesis . Thermostability of the mutant was much lower than the wild-type enzyme, which suggests that the carbohydrate chain of AmylA is important for the enzyme stability. The differences in reaction properties between the wild type enzyme and the mutant suggest that the carbohydrate chain of AmylA significantly affects the hydrolysis efficiency and the substrate recognition of AmylA for soluble starch . From the comparison of amino acid sequence of AmylA to those of other α-amylases whose 3D structures were clarified, the carbohydrate chain of A mylA is suggested to be positioned on the surface near the active cleft. Therefore, direct interactions between the carbohydrate chain and the substrate might affect the hydrolysis efficiency and substrate recognition.

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By use of the emulsions composed of innert gelatin and octahedral or cubic silver bromide grains (about O.7pt), effects of sulfur and gold sens, itizations have been examined on the emission processes at liquid N2 temperature and photographic processes of the silver bromide grains. Both sulfur and geld sensitizations increased the photographic speeds, and decreased the emission intensities of both octahedral and cubic grains. This result has been interpreted by the view that both sulfur and gold sensitizations provide electron traps at the grain surfaces.

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It has long been known that ungerminated barley contains only β-amylase while α-amylase appears at germination and that there are two types of β-amylases, active and inactive. Many scientists have so far paid much attention to the activation mechanism of the inactive β-amylase. However, their papers were found to have some problems left unresolved. One of the main reasons seems to be due to the failure to isolate inactive β-amylase in a native state and no experiments on it in vitro. Therefore, I attempted to isolate salt-soluble inactive β-amylase and two types of inactive enzymes, heteropolymer type (MW. 280, 000) and homopolymer type (MW. 160, 000) were isolated. From the activation of these enzymes in vitro, it was shown that both reductive cleavage of disulfide bonds and proteolytic cleavage of peptide bonds are necessary for the complete activation of all the types of inactive β-amylases in ungerminated barley. On the other hand, the activation of these amylases during germination was found to proceed mainly through protein disulfide reductase and malt proteinase. On the basis of these results, it was concluded that active β-amylase synthesized in the early stages of barley ripening is polymerized by disulfide bonds and/or peptide bonds into inactive enzymes in the later stages of ripening and that, during germination, the inactive enzymes are again depolymerized into active β-amylase which hydrolyzes starch with α-amylase to supply energy for germination. It is interesting that the reversible reactions between the sulfhydryl and disulfide groups are concerned with the activation and inactivation of β-amylase in barley. In the 1970s the distribution of fl-amylase was reported in microorganisms. We also isolated several microorganisms producing β-amylase from soil. Bacillus cereus BQ10, one of the isolates, was treated by UV irradiation for enhancement of β-amylase productivity. The BQ10-S1, UV-mutant was found to produce about 30 times more β-amylase than the wild strain. Furthermore, a rifampin-resistant, asporogenous mutant, BQ10-S1 Spo-, was obtained by NTG treatment. The amount of β-amylase produced by this asporo-genous mutant reached about 500 times that of the wild strain. The molecular weight of the enzyme was 60, 000 and the optimum pH and temperature were around neutral pH and 55°C. The gene of β-amylase was isolated and cloned in E. coli, and all the base sequences and amino acid sequences were determined. The homology of the amino acid sequences between other plants and microbial β-amylases was compared. About 50% homology was found among those from B. polymyxa, B. circulars, Cl. thermosulfurogenes, etc., and about 30% among those of soybean, barley, sweet potato, etc. Amino acid residues at the catalytic site of β-amylase, which have long remained unelucidated, were examined with B. cereus crystallized β-amylase in enzymatic and X-ray crystallographic studies and found to be not sulfhydryl but two glutamic acid residues. The hydrolytic activity of raw starch by BQ10-S1 Spo-was also found to be due to a "starch-binding domain (SB)" which was not found in plant β-amylases.

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We have established some novel synthetic transformations of simple sugars into useful oligosaccharides utilizing the transglycosylation of glycosidases in aqueous -organic solvent system. p-Nitrophenyl penta-N-acetyl-β-chitopentaoside, which is useful as a novel substrate for lysozyme assay, was efficiently synthesized through a lysozyme-catalyzed transglycosylation on penta-N-acetylchitopentaose and p-nitrophenyl β-N-acetylglucosaminide. In this case, the use of an aqueous-DMSO system in the reaction not only ensured the solubility of chromogenic substrate, but also resulted in the high yield of the desired compound. This concept was introduced for the preparation of p-nitrophenyl α-maltopentaoside from maltopentaose and p-nitrophenyl α-Dglucoside by the use of maltotetraose-forming amylase from Pseudomonas stutzeri. Furthermore, p-nitrophenyl N-acetyl-Iactosaminide and p-nitrophenyl N-acetyl -allolactosaminide were regioselectively synthesized from lactose and p-nitrophenyl N acetylglucosaminide by using the transglycosylation of β-D-galactosidase from Bacillus circulars by controlling the concentration of acetonitrile in the reaction system, respectively.

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The gene coding for the maltopentaose (G₅)-forming enzyme of Pseudonaonas sp. KO-8940 was cloned into Escherichia coli and its nucleotides sequenced. It was found to have a long open reading frame composed of 1842 by that encoded 614 amino acid residues for a secretory precursor polypeptide including the typical signal sequence with an NH₂-terminal. In the deduced primary structure of this enzyme, a high degree of homology to four regions conserved by many α-amylases was found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases. The G₅-forming enzyme was produced in large amount (52.7 IU/ml, 0.1 g/l) in E. coli under the tac promoter. This result showed that the G₅-forming enzyme can be produced in E. coli carrying this enzyme gene expression vector at levels up to 6 times greater than the native production system found in P, sp. KO-8940.

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　鶏卵卵黄由来のシアリルオリゴ糖画分の学習能力に与える影響を調べた.鶏卵卵黄から調製したシアリルオリゴ糖画分を乳飲み子のラットに14日齢から21日齢の期間,経口投与した.42日齢から49日齢の8日間迷路を用いてゴールまでの到達率と到達時間を測定した.その結果,シアリルオリゴ糖画分投与群は,コントロール群と比べゴールまでの到達率が高く,到達時間もコントロール群と比べ有意に短時間であった.今回の実験から新生児期のシアリルオリゴ糖の摂取は,新生児の学習能力の発達にとって重要な成分であると考えられた.

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出発原料(兼発熱体)としてカーボンブラックを用い,高圧溶融法で黒鉛単結晶を生成した。カーボンブラックにはごく微量のFe,CaとTiが含有されていた。ピストン,シリンダー型高圧装置を用いた。その詳細については前脚に報告した。加圧下での高温度補正が困難であるために,温度のかわりに入力電力(W)で表示した。発熱体表面に黒鉛単結晶が,2kb下で1020W以上の条件で生成した。最大のものは約12.O×10.0mm2であり,厚さは1mmの数分の一である。その生成機構については,パイロフィライト中に含まれる元素が重要な役割を演じていることがかなり確実のようである。発熱体内部では,2kb下で1400W以上の二条件で黒鉛単結晶が生成した。最大のものは120×0.85mm2くらいであり,金属光沢を有し,よく発達した稜をもっていた。生成黒鉛単結晶上に双晶境界のクラックが観察された。その双晶の角度は16.4 と11.1 であった。ある実験条件では発熱体の中心付近に黒鉛球状晶が生成した。その最大のものは直径約1.35mmであった。小さい個々の黒鉛単結晶の基底面は球状結晶の円周に平行に配向していた。X線回折結果から,生成黒鉛単結晶のC0値は天然産のものとほぼ同程度であった。

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