The dietary response of mucosal ornithine decarboxylase (ODC) in meal-fed and refed rats was investigated for each quarter of the small intestine. The intake of a normal powdered diet caused a considerable postprandial increase in the ODC activity in the mid two quarters, while the intragastric infusion of an aqueous solution containing amino acids and glucose elevated the ODC activity to a large extent in the proximal quarter as well as in its contiguous mid quarter. Such responsiveness was almost completely depressed by previous α-difluoromethylornithine (DFMO) administration. Mucosal scrapings from the small intestine of rats with ODC depression did not significantly differ from those without ODC depression as to their [³H]thymidine-, [³H]uridine-, and [³H]leucine-incorporating capacity into the DNA, RNA, and protein fractions, respectively, at various postprandial times. On the other hand, putrescine was found to plentifully accumulate in the ileal lumen within a few hours after the start of eating, suggesting that the ODC depression would thereby have been compensated. To investigate this further, putrescine-uptake experiments were carried out with both everted sacs and in situ loops of the small intestine. It is assumed from the results that [¹⁴C]putrescine was transported a little more across the ileal wall than across the duodenal or jejunal wall by a Na⁺- or energy-independent mechanism, and that [¹⁴C]putrescine taken up from the lumen distributed itself within the whole body.

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The growth responses of a variety of human intestinal bacteria to partially hydrolyzed guar gum (PHGG) were investigated in vitro and in vivo. In an in vitro experiment, PHGG moderately enhanced growth of some bacterial strains including Bacteroides ovatus, Clostridium coccoides, C butyricum, and Peptostreptococcus productus. Effects of PHGG intake (7g/volunteer, 3 times per day, for 14 days) on fecal microflora, bacterial metabolites, and pH were investigated using nine healthy human volunteers. The count of Bifidobacterium spp. and the percentage of these species in the total count increased significantly during the PHGG intake periods. Among the acid-forming bacteria, Lactobacillus spp. also increased. The fecal pH and fecal bacterial metabolites such as β-glucuronidase activity, putrefactve products, and ammonia content were significantly decreased by PHGG intake. Two weeks after the end of PHGG intake, the bacterial counts and their biological manifestations appeared to return to the former state.

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New bis-piperazine-type pH buffer agents were synthesized and their buffering properties were evaluated. The compounds proved to have two-fold larger pH buffering ability than 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), a Good's buffer traditionally used to control the pH value of culture media. Human-human hybridoma HB4C5 cells were cultured in a serum-free medium containing these buffer agents. The cell growth and antibody production, using 1, 2-N, N'-bis[N", N"'-di(2-sulfonoethyl)piper-azinyl]ethane, were greater than when HEPES.

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Casein phosphopeptide (CPPcm), which inhibits the precipitation of calcium phosphate in the intestines, was prepared as a CPPcm-alcium phosphate complex from casein micelles (casein-calcium phosphate conjugates). CPPcm was eluted as seven peaks from a Q-sepharose FF column, the major peaks being identified and agreeing with those of the phosphopeptides (CPPcn) from acid-precipitated casein. The major components of CPPcm were α-<s1>-CN-5P(f59-79) and β-CN-4P(f1-25), CPPcm containing more α-<s1>-CN-5P (f59-79) than CPPcn. The complexes of CPPcm and calcium phosphate contained twice the quantity of calcium phosphate as those of CPPcn.

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The influence of linear alcohols on the formation of inclusion complexes between d-limonene and α-, β-, or γ-cyclodextrin has been examined at a low water content using a micro-aqueous method. In the presence of alcohols, particularly methanol and ethanol, the formation of inclusion complexes between d-limonene and β- or γ-cyclodextrin is enhanced. The alcohol included in the cyclodextrin at the low water content was replaced with d-limonene as the water content increased. This implies that these alcohols would behave like water and activate the inclusion reaction between d-limonene and β- or γ-cyclodextrin. On the contrary, the formation of -limonene complexes with α-cyclodextrin is inhibited by the presence of alcohols except for methanol. By means of the apparent water content, the amount of d-limonene complex in β- and γ-cyclodextrin correlated well with a single sigmoid curve.

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Products of strawberry jam prepared by a high hydrostatic pressure-processing method and a conventional heat-processing method were examined with regard to the changes of quality such as volatile flavor components, anthocyanins, browning index, furfural, sucrose contents, and vitamin C contents during storage at 5°C and 25°C for 1-3 months. The quality of pressure-processed jam immediately after manufacturing was superior to that of heat-processed jam in preserving the fresh flavor and natural color of raw fruits. The superior quality was maintained under low temperature storage for two to three months. However, room temperature storage resulted in rapid deterioration including off-flavor, discoloration, browning, and decomposition of sucrose and vitamin C. So, the commercial value of pressure-processed jam decreased within a brief period. On the other hand, heat-processed jam did not change in quality under room temperature storage for three months. It is supposed that dissolved oxygen and enzyme systems retained are the causative factors of rapid deterioration in pressure-processed jam.

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Three α-glucosidases which passed under the names of transglucosidase (from Aspergillus niger), maltase (from Brewers yeast), and isomaltase (from Bakers yeast) for reasons of their substrate specificities and transfer actions, were purified to electrophoretically pure states. These purified α-glucosidases were made uniform in the hydrolyzing activities using p-nitrophenyl 2-glucopWanoside (α-p-NPG) and were reacted with p-nitrophenyl α-xylopyranoside (α-p-NPX) or isoprimeverose (xylopyranosyl-α-1, 6-glucopyranose), which are typicalsubstrates of α-xylosidase. Only Asp. niger α-glucosidase among them hydrolyzed α-p-NPX and isoprimeverose. Further the substrate specificities of three α-glucosidases and two α-xylosidases (I and II from Asp. flavus MO-5) were investigated on maltose, isomaltose, α-p-NPG, isoprimeverose, and α-p-NPX in detail, and kinetic parameters [K_m, V_max, and molecular activity (k_o)] were estimated and compared with each other. In the comparison of kinetic parameters, Asp. niger α-glucosidase showed a broad specificity, that is, containing isoprimeverose in addition to maltose, isomaltose, and α-p-NPG. Though this enzyme barely hydrolyzed α-p-NPX too, the velocity was very slow. Though both yeast α-glucosidases barely hydrolyzed α-p-NPX or isoprimeverose too, these substrates were not good for yeast enzymes. On the other hand, two α-xylosidases showed narrow speciticities, such that the substrates except for α-p-NPX and isoprimeverose were not hydrolyzed at all. The action on isoprimeverose by Asp. niger α-glucosidase was completely the same as that on isomaltose at optimum pH, optimum temperature, inhibition pattern of hydrolyzing activity by 1-deoxynojirimycin, and transfer action pattern. Accordingly, we interpret these results as indicating that the hydrolyzations of isomaltose and isoprimeverose by Asp. niger α-glucosidase were catalyzed at the same active site. Asp. niger enzyme that has both α-glucosidase activity and α-xylosidase activity was shown to be classified in a middle position between α-glucosidase and α-xylosidase.

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Cytidine-producing mutants were derived from Bacillus subtilis No. 122. Strain CD-300, a cytidine deaminase deficient derivative of strain No. 122, accumulated 0. 1mg/ml cytidine in the culture broth. Growth of CD-300 was inhibited by 6-azauracil and 5-fluorocytidine. The inhibition by 6-azauracil was reversed by added uracil, and that by 5-fluorocytidine was by cytidine. 6-Azauracil-resistant mutants were derived from CD-300. Most of the mutants accumulated both uracil and uridine. A representative strain, No. 229, accumulated 6. 2mg/ml uracil and 1. 0mg/ml uridine in the culture. Subsequently, 5-fluorocytidine-resistant mutants were derived from No. 229. Among them, strain No. 344, which was resistant to 500pg/ml 5-fluorocytidine, accumulated 10. 4mg/ml cytidine in the culture broth.

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Fractionation of several kinds of membranes from the taste organ, circumvallate papillae, of bovine has been done to investigate localization of the umami substance binding site. Monosodium glutamate (MSG) and nucleotides are known as umami substances. The membrane fractions obtained from a series of centrifugation were examined for radioactive MSG binding activities by the ligand-binding methods, indicating a synergistic effect between MSG and nucleotides. They were also measured for activities of the marker enzymes. The membrane fraction that seemed to be rich in plasma membrane, on the basis of the marker enzyme activities, had specific MSG binding activity. These results suggest that umami substances bound to the plasma membrane of circumvallate papillae.

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To develop a model system for silage fermentation that is simple, rapid, and repeatable by using storable plant materials, we made several experiments of simulated silage fermentation in culture tubes. In these experiments, we used milled alfalfa hay cubes (AHC) mixed with certain amounts of water and glucose (AHC medium) and a lactic acid bacterium (LAB) was cultivated together with or without a butyric acid bacterium (BAB) and a coliform bacterium (CFB) under anaerobic conditions. The pure culture of LAB in AHC medium produced lactic acid within 24h of incubation. The mixed culture of CFB with LAB and BAB promoted butyric fermentation. Butyric acid was produced only with high temperature (more than 30°C), low dry matter content of AHC medium (less than 20%), and prevention of lowering the medium pH. Low soluble sugar content of the medium (0. 35%) or smaller inoculum size of LAB (less than 10⁶ cfu/g)than that of CFB (10⁷cfu/g) was effective for the prevention of lowering the medium pH. This modelsystem seemed to be useful for screening of LAB as a silage inoculant.

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We screened of lactic acid bacteria (LAB) for silage inoculants by using a model system for ensilage. For 16 strains of LAB selected as having high lactic acid production in a liquid culture medium, we examined lactic acid production in the model system. Among them, strains 5-6 and 5-29 had the highest lactic acid production due to higher growth rates than those of the other strains and strain 1-1O was more acid-tolerant than the other strains. The specific growth rate of strain 1-10 was lower than those of strains 5-6 and 5-29. Strain 1-10 was identified as Lactobacillus casei. Strains 5-6 and 5-29 were identified as Lactobacillus plan tarum.

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Lymph fistula rats were continuously infused with emulsions containing diacylglycerol consisting of 1, 3-species (65. 6%), 1(or 3), 2-species (32. 6%), and triacylglycerol (rapeseed oil) at the rate of 3ml/h for 1h through a cannula inserted into the stomach. The lymph fluids were collected every hour for 5h after starting the infusion of the lipid emulsions, and the lymph chylomicrons were isolated, purified and analyzed. Test emulsions were prepared to provide the same amount of fatty acids (144mg/h) as that in these acylglycerols. The rates for triacylglycerol transport at 2-3 h and for cholesterol transport by chylomicrons at 2-3h and 3-4h of the experimental period in rats infused with the diacylglycerol emulsion were significantly lower than the corresponding values in the rats infused with the triacylglycerol emulsion. As a consequence, the cumulative value for triacylglycerol transport at the end of the experimental period in rats infused with the diacylglycerol emulsion was significantly lower than the corresponding value in the rats infused with the triacylglycerol emulsion. In addition, cumulative values for cholesterol transport from 3h to the end of the experimental period were significantly lower in the former than in the latter. There was no difference in the total fatty acid compositon of chylomicron-triacylgIycerol between the rats receiving the triacylglycerol and diacylglycerol emulsions. However, a considerable difference existed in the fatty acid composition at the 2-position of triacylglycerol between the two groups of rats. Thus, intragastric infusion of diacylglycerol mainly consisting of the 1, 3-species compared to triacylglycerol not only altered the rate of lipid transport by lymph chylomicrons but also altered the structure of the triacylglycerol moiety in the rats.

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Mutagenic activity and DNA intercalation were examined for 9-aminoacridine (9-AA) and its deriva-tives. Introduction of a nitro group into the 9-AA molecule was found to enhance the activity enormously as was detected by the Ames test. Acetylation of amino group at 9-position of acridine ring inhibited the intercalation, the frameshift activity disappearing. Rat liver S9 converted 9-AA metabolically to 9-amino-2-hydroxyacridine.

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Based on the partial amino acid sequences of a protein disulfide isomerase (PDI) from Humicola insolens, two primers were synthesized for reverse transcriptase mediated ploymerase chain reaction (RT-PCR) of a fungal RNA. A 0. 2-kbp fragment around the consensus sequence of PDIs was obtained and used as a probe for screening a fungal cDNA library. A cDNA clone of PDI from H. insolens was isolated and encoded a polypeptide consisting of 505 amino acids, which was characterized by a N-terminal signal sequence composed of 20 amino acids, a consensus sequence (WCGHCK) at two positions, and a C-terminal endoplasmic reticulum retention signal (HDEL). Bacillus brevis harboring an expression plasmid bearing the fungal PDI cDNA was prepared and its culture supernatant showed a signiticant PDI activity. This indicates that glycosylation of a fungal PDI is not essential for the enzymatic activity related to an interchange of disulfide bonds.

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An unicellular green alga identified as Lobosphaera sp. by morphological observations was selected as a source of trehalase. The alga grew well heterotrophically and produced intracellular trehalase using Polypepton, yeast extract, and glycerol as nutrients. The enzyme was highly puritied by ammonium sulfate fractionation, column chromatography on DEAE-Toyopearl, Sepharose CL-4B, and SP-Toyopearl. The molecular mass was estimated to be 400kDa by gel filtration. SDS-PAGE indicated that the enzyme consisted of two subunits with a molecular mass range of 180-220kDa and it contained carbohydrates. The enzyme was most active at pH 5. 5 and at 65°C and stable between pH 4-9 and below 65°C. Fe³⁺inactivated the enzyme. Sucrose was a competitive inhibitor with a K_i of 7. 5mM. The enzyme specifically hydrolyzed trehalase with a Km of 0. 6mM.

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A purified trehalase from Lobosphaera sp. catalyzed the condensation not only of D-glucose but also of 2-deoxy-D-glucose, yielding α, α'-trehalose and 2, 2'-dideoxy-α, α'-trehalose, respectively. Simultaneous incubation of D-glucose and 2-deoxy-D-glucose resulted in the formation of 2-deoxy-α, α'-trehalose in addition to the above disaccharides. The reaction under various conditions showed that the enzyme synthesized dideoxy-trehalose (yield, 16%) more than trehalose (5%). Monodeoxy-trehalose was produced most effectively in the reaction of D-glucose and 2-deoxy-D-glucose in a weight ratio of 2 : 5, and at this ratio, 6%, of the total weight of two substrates was converted to the product.

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Thermolysin-catalyzed peptide synthesis using V(benzyloxycarbonyl)-L-phenylalanine (Z-Phe) and L-phenylalanine methyl ester (Phe-OMe) as substrates was done mainly in a water-organic one phase solvent system. The organic solvent content used was less than the saturation concentration in buffer. With organic solvents with high log P values, the enzymatic activity increased as the organic solvent content increased ; but further increases in the organic solvent content decreased the enzymatic activity, showing an "organic activity" profile. On the other hand, with organic solvents of low log P values, the enzymatic reaction was inhibited even by the initial addition of organic solvents. When a correlation between maximum activities and logP values or Hildebrand solubility parameters was investigated, a linear correlation was obtained among the same category of organic solvents, but not between categories. This suggests that the direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvents.

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Repeated selection of root lines that were highly productive for scopolamine was investigated for Agrobacterium rhizogenes-transformed and untransformed roots of Duboisia myoporoides. Lines highly productive for scopolamine could be selected by repeated selection of transformed roots, but not by repeated selection of untransformed ones. In transformed root cultures, the scopolamine content of the highest scopolamine-producing root line after 8 selections was 3. 2% dry weight (DW) as compared to 0. 15% DW for the parent line. The total alkaloid contents and scopolamine/hyoscyamine ratio also increased. Repeated selection decreased growth from 7. 0 to 2. 2g/liter, but there was no correlation between growth and scopolamine content in the selected transformed root cultures. This enhancement of scopolamine content and decrease of growth appears to be caused by the heterogeneous nature of the transformed roots.

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A circulation culture system was established for the high density root culture of Duboisia roots. It consists of a vessel for root culture, an aeration tube, a medium reservoir, and a peristaltic pump. Medium saturated with pure oxygen was circulated continuously through the culture vessel and the medium reservoir, and was pumped back into the aeration tube by the pump. Duboisia roots could be cultured at densities of up to 120g dry weight (DW) dm^-3 with no decrease in scopolamine content, this density being about 20times the amount that can be used in ordinary flask culture. The final scopolamine yield at the end of 3 weeks was 1350mgdm^-3.

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We cloned the genes from Corynebacterium sp. strain N-1074, for two halohydrin hydrogen-halide-lyase isoenzymes (hheA and hheB), which are involved in the transformation of alpha, beta-halohydrin into the corresponding epoxide and the reverse reaction. The nucleotide sequences of 1057 base pairs including the hheA gene and of 1130 base pairs including the hheB gene were analyzed. The predicted amino acid sequence of the hheA gene product consisted of 244 residues, and the calculated molecular weight was 26, 465. The hheB gene had two sets of potential ribosome binding site and a possible initiation codon. The predicted amino acid sequences of the gene products consisted of 235 and 227 residues, and the calculated molecular weights were 26, 179 and 25, 236, respectively. Site-directed mutagenesis experiments strongly suggested dual translational initiation in the hheB gene. Comparison of the predicted amino acid sequences for the hheA and hheB genes found signiticant homology only in the carboxyl terminal region. An analysis of the upstream regions of the both hhe genes suggested the presence of putative epoxide hydrolase genes, which might be involved in further degradation of epoxide compounds.

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An active cell-free translation system was prepared from Brevibacterium lactofermentum, a Grampositive bacteria used in molecular cloning and protein expression. The system contained high speed postribosomal supernatant (S 370), purified ribosomes and a tRNA mixture from Escherichia coli, and synthesized polyuridylic acid-directed polyphenylalanine once optimized for mono and divalent ions, time, and temperature. The translation system was evaluated for sensitivity to several translational inhibitors including several N-glycosidase ribosome-inactivating proteins (RIPs) isolated from plants. The pattern of inhibition by RIPs resembled that observed recently for Gram-negative bacteria such as Escherichia coli and Agrobacterium tumefaciens [Girbes et al., J. Bacteriol., 175, 6721-6724 (1993)]. A typical inhibitory type 1 RIP such as crotin 2 promoted depurination of the rRNA, which upon treatment with acid aniline released a fragment of approximately 230 nucleotides. On these grounds, we propose that bacterial ribosome sensitivity to plant RIPs depends on the bacterial ribosome-specific presence of protein recognition domains in the RIP present only in some RIP but not in others.

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α-Glucuronidase is a very important enzyme for the complete hydrolysis of plant hemicellulose, but the substrate specificity of the enzyme has not previously been reported. In this connection, the three regioisomers of O-(α-D-glucopyranosyluronic acid)-D-xylose (aldobiouronic acid), 2-O-(α-D-glucopy-ranosyluronic acid)-D-xylose (13), 3-O-(α-D-glucopyranosyluronic acid)-D-xylose (14), and 4-O-(α-D-glucopyranosyluronic acid)-D-xylose (15), were stereoselectively synthesized to clarify the substrate specificity.

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An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field ele-ctrophoresis. Seven chromosomal bands were found. With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7. 0, 5. 2, 5. 0, 4. 5, 4. 0, 3. 7, and 2. 8 megabase pairs (Mbp). The chromosomal DNA bands were identified with use of 13 cloned genes including ribosomal DNA. The intensity of ethidium staining and the results of Southern blotting with 100 random clones isolated from A. oryzae suggested that the smallest band migrated as doublet and that the total genome size was approximately 35Mbp.

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The specificity of five mouse monoclonal antibodies to okadaic acid was studied for use in an enzyme-linked immunosorbent assay of okadaic acid and its analogs. OA8-2 and OA22-22 antibodies (IgG-<2a>-K), which bind more strongly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 than to okaic acid or 7-O-palmitoyl-okadaic acid in 50% aqueous methanol, were useful in the detection of dinophysistoxins-1 and -3. OA10-8 (IgG₁-K), which binds more strongly to 7-O-palmitoyl-okadaic acid and 7-O-palmitoyl-dinophysistoxin-1 than to okadaic or dinophysistoxin-1 in 50% aqueous methanol, was useful in the detection of dinophysistoxin-3. OA423-3 (IgG₁-K), which binds weakly to dinophysistoxin-1 and 7-O-palmitoyl-dinophysistoxin-1 in 20% aqueous methanol, was useful in the selective detection of okadaic acid. OA958-2(IgG₁-K), which binds with equal strength to each of the four toxins in methanol, was useful in the detection of all okadaic acid analogs, and the minimum detectable concentration was 30ng/ml. OA423-3 and OA958-2 retained their binding ability in 50% acetone, ethyl ether, or benzene in methanol.

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cDNA clones coding cecropins A and B were isolated from a cDNA library constructed from the fat body of immunized Bombyx mori larvae. The cloned cDNAs had an open reading frame of 63 amino acids, indicating the primary translated peptides were processed to form mature cecropins of 35 amino acid residues. The homology in the coding regions of cecropins A and B was 73%. In immunized fat body, the expression of both cecropin A and B genes reached the maximal level 5h after the injection of soluble peptidogIycan, and the high level was maintained until 9h after immunization. The cecropin A and B genes were expressed at high levels in fat body and hemocytes, at lower but significant levels in malpighian tube, slightly in midgut, and none in silk gland.

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The 7-O-[2-O-(α-L-Rhamnopyranosyl)-β-L-quinovoside] of naringenin and of hesPeretin, and their dihydrochalcone (DHC) derivatives were synthesized by the method of Koenigs-Knorr (Ag₂CO₃ and quinoline). The reaction of TMS ethers of naringenin and of hesperetin with each of the α-acetofluoro derivatives of D-glucose, L-rhamnose, 2-O-(α-L-rhamnopyranosyl)-L-rhamnose, and 2-O-(α-L-rhamnopy-ranosyl)-D-glucose (neohesperidose), using boron trifluoride etherate as an activator, yielded coupling prod-ucts which, after deprotection, gave naringenin 4'-O-β-D-glucoside, naringenin 4'-O-α-L-rhamnoside, narin-genin 4'-0-[2-0-(α-L-rhamnopyranosyl)-2-L-rhamnoside], hesperetin 3'-0-[2-0-(α-L-rhamnopyranosyl)-2-L-rhamnoside], and naringenin 4'-0-β-neohesperidoside, respectively. Catalytic hydrogenation of these flavanones gave the corresponding DHC derivatives. Hesperetin DHC 4'-O-[2-O-(α-L-rhamnopy-ranosyl-β-L-quinovoside] was 30O times sweeter than sucrose, while the other compounds were bitter or tasteless.

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A latent form, mostly soluble, of polyphenoloxidase of La France pear fruit (Pyrus communis) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography with DEAE-Sephadex A-25 and then DEAE-Toyopearl 650M, followed by gel permeation chromatography with Toyopearl HW-55s. The addition of 10% glycerol to the eluting buffer was needed for purification. The purified latent enzyme seemed to be a monomeric protein ; the molecular weight was estimated to be 70, 000 by gel permeation chromatography and 65, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was activated by pressurization at 400MPa or higher or by treatment with SDS. The highest activity was obtained by pressurization at 600MPa and 20°C for 6h.

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The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1, 536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56, 210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Taq prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases. However, a His-Glu-X-X-His sequence, which is a conserved sequence in the ac-tive site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Taq is a novel type of zinc-dependent metallo-carboxypeptidase.

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Xylanase A encoded by the C1ostridium stercorarium F-9 xynA gene was puritied to homogeneity from a recombinant clone of Escherichia coli. The N-terminal amino acid sequence and molecular weight (54, 000) estimated by SDS-PAGE of the purified enzyme were consistent with those deduced from the nucleotide sequence [Biosci. Biotech. Biochem., 57, 273-277 (1993)]. A xylanase was also purified to homogeneity from a culture supernatant of C. stercorarium F-9. Its N-terminal amino acid sequence, molecular weight, and enzymatic properties were quite in agreement with those of the recombinant enzyme, indicating that the xynA gene was predominantly expressed as a xylanase gene in C. stercorarium F-9. The purified enzyme hydrolyzed xylotriose to yield xylobiose and xylose while it was less active toward xylobiose. It was optimally active at 75°C and pH 7. 0. K_m and V_max were estimated to be 1. 9mg/ml and 2. 8μmol of xylose equivalent/min/μg for oat spelt xylan, respectively.

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Oryzacystatin (OC) is a proteinaceous cysteine proteinase inhibitor involved in the biodefense of rice seeds. To create transgenic rice plants with increased OC activity, we introduced an OC expressing vector into rice protoplasts and obtained transformed calli. The expression vector contained a bacterial inaA DNA fragment in the 3'-noncoding region as a tag to distinguish the introduced DNA from the intrinsic OC gene. The OC vector and a selection marker gene conferring hygromycin resistance were used together to transfect into rice protoplasts. A number of hygromycin-resistant calli were obtained and studied by polymerase chain reaction and genomic Southern blotting to find if the exogenous OC gene had been integrated. The calli were studied by northern blotting as well to examine mRNA expression. The results showed that integration dnd expression of the introduced OC gene occurred in 51% and 27%, respectively, of 156 subcultures from 15 hygromycin-resistant calli. As a final step, transgenic rice plants were regenerated from the calli expressing OC. Leaves and seeds from the plants had higher OC activities than those from nontransgenic Plants.

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Seven out of 20 assayed picrotoxane terpenoids inhibited specific binding of [³⁵S] t-butylbicyclophosphorothionate to rat brain membranes, with IC₅₀ values of 0. 0075-6. 0μM. Picrodendrin Q was identified as the most potent compound, being about 27-fold more potent than picrotoxinin. The high activity of picrodendrins A, M, and Q suggests the significance of the spiro α-ethylidene γ-lactone moiety for the interaction of picrodendrins with the GABAA receptor-coupled picrotoxinin binding site.

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Ether analogues of a plant growth regulator isolated from Botrytis squamosa, (2S, 2'S)- and (2S, 3'R)-dimethyl 3-phenyl-2, 2'-oxydipro-pionate (1), were prepared by the Williamson synthesis, and the two diastereomers were separated. The absolute configurations of both diastereomers were determined by chemical transformation. The promoting effect of both synthesize analogues on lettuce seedling growth was similar to that of the natural plant growth regulator.

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Trimeresurus flavoviridis venom gland phospholipase A₂ (PLA₂) genes pgPLA 1a and pgPLA 2a encode Asp-49-PLA₂ and genes pgPLA 1b and pgPLA 2b encode an isozyme of Asp-49-PLA₂. Polymorphisms were found in pairs of pgPLA 1a and pgPLA 2a and of pgPLA 1b and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated.

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The ATP level was exclusively increased among the nucleotides in Escherichia coli under osmotic stress, concomitant with a decrease of guanosine phosphates levels. The profile of ATP formation was different from that resulting from chemicals. E. coli contains a specific system to enhance the ATP level and it might be possible that the source of the increased ATP is guanosine phosphates.

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Freshly isolated hepatocytes were cultured on an EHS-gel prepared from EHS-tumor, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), and type I collagen (TIC). Hepatocytes on EHS-gel showed a spherical shape and much more strongly maintained the inducible expression of cytochrome P-450 genes which were lost on PVLA and TIC. Further, the expression of liver-specific genes were maintained on EHS gel at the highest level, and then higher on PVLA than TIC.

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The fungus SKB-1152 bleaches oxygen-alkaline treated hard wood kraft pulp (OKP) rapidly. In the initial phase of fungal treatment, maximum production of manganese peroxidase (MnP) was observed. The filtrate from a 1-day fungal treatment could bleach OKP when manganese, glucose, and glucose oxidase were added. A possible role of MnP in the initial fungal bleaching process is suggested.

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Both growth and theanine accumulation in tea callus cultures were improved by the combination of 4mg/liter benzyladenine and 2mg/liter indol-3-butyric acid, but were strongly inhibited by the addition of 2, 4-dichlorophenoxyacetic acid. The optimum initial concentration of carbon source was 30g/liter sucrose. Upon the addition of more than 30g/liter of sucrose, the callus fresh weight was increased, but the theanine formation was not improved.

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The amino acid residues within potential active sites of monellin were replaced. The replacement of Ile^B6, Asp^B7, or Ile^B8 by different amino acids resulted in a complete or marked loss of sweetness, while the replacement of Tyr^A13 or Asp^A16 by other amino acids did not substantially reduce the sweetness. The replacement of each Lys residue by L-2-aminohexanoic acid significantly reduced the sweetness potency, but did not completely remove the sweetness. An analysis of synthetic analogs by CD showed that the major structural features of monellin were not significantly altered. These findings suggest that Ile^B6, Asp^B7, and Ile^B8 are responsible for eliciting a sweet taste, and that one or more basic residues may also be important for sweetness.

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The biosyntheses of sescandelin (1) and sescandelin B (2) were studied by feeding of [¹³C]labelled precursors to Sesquicillum candelabrum. The labelling pattern of those compounds enriched from the [1-¹³C], [2-¹³C], and [1, 2-¹²C]acetates, and from the [¹³C]formate showed that both compounds were derived from a pentaketide chain and a C₁ unit. The isocoumarin skeleton of 1 and 2 is considered to have been formed by the cyclization of a pentaketide chain and a C₁ unit, and of a pentaketide, respectively.

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The existence of "plastid initials" was not discovered until recently. Such organelles can be extracted from cortical cells of poplar by enzymatic digestion of the excised tissues. As judged from electron micrographs, the occurrence of the novel bodies in a free state in the cortical cells has been confirmed.

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The anthocyanin production of cultured Euphorbia millii cells using envelope-shaped film culture vessels increased with the decrease of film thickness. The optimum temperature for anthocyanin production under the illumination was at 22°C. The anthocyanin production was increased by 2 times with mild agitation.

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Three glycosides, 6-O-β-D-xylopyranosyl-β-D-glucopyranosides (β-primeverosides) of the aroma constituents, linalool, 2-phenyletha-nol, and benzyl alcohol, were isolated as aroma precursors from the tea leaves (Camellia sinensis var. sinensis cv. Shuixian and Maoxie, cultivars for oolong tea). The isolation was guided by acid or enzymatic hydrolysis, and subsequent GC and GC-MS analyses. The linalyl glycoside is the first example of naturally occurring (S)-linalylp-βprimeveroside.

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The active site of α-glucosidase from Mucor javanicus IFO 4570 was investigated by kinetic studies. Competition between maltose and soluble starch, and linearity of Lineweaver-Burk plots for the mixed substrates were observed. The dependence of the apparent maximum velocities agreed with those predicted for a single active site mechanism. These results suggest that the enzyme hydrolyzes maltose and soluble starch at a single active site.

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In the presence of silver carbonate, primary and secondary alcohols such as γ-hydroxybutyrophenone, 17_α-21-dihydroxy-4-pregnene-3, 11, 20-trione and 11_α-hydroxy-17_α-methyltestosterone react with alkyl halides in N, N-dimethylformamide under mild reaction conditions to give the corresponding unsymmetrical dialkylcarbonates in good yields.

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A 5'-flanking region (- 2024 to +61) of the guinea pig liver transglutaminase gene and some 5'-deletion mutants were tested for promoter activity in human hepatoblastoma HepG2 cells treated with interleukin-6 (IL-6) by an assay of the transient expression of the chloramphenicol acetyltransferase reporter gene. The promoter activity of the 5'-flanking region introduced into the HepG2 cells was increased by IL-6.

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A novel sterol with a heptanorergostane skeleton was isolated from the neutral fraction of Lactarius volemus, together with two known sterols. The structures of these compounds were determined by chemical transformation of ergocalciferol and by spectroscopic methods.

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A screening test was undertaken to isolate microorganisms that produced dipeptidyl aminopeptidase. The hydrolytic activity toward alanyl-phenylalanine p-nitroanilide was found in a culture filtrate of a actinomyces strain (WM-23), newly isolated from a soil sample. The enzyme (WM-23 dipeptidyl aminopeptidase) was isolated from the culture filtrate as a homogeneous preparation. The WM-23enzyme, inhibited by phenylmethylsulfonyl fluoride, may be classitied to mammalian dipeptidyl aminopeptidase II. The enzymatic characteristics were investigated.

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Four triacylglyceride hydroperoxides were synthesized by DCC-mediated esterification of a dimethylperketal of 13-hydroperoxy-octadecadienoic acid with glycerides, in which one or two linoleoyl groups were linked, and by final removal of the protective group with a mixture of THF, acetic acid and water.

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