Syntheses of [1-¹³C]-2-butenedinitrile in a one-pot process and of pyridoxines regiospecifically multi-labeled with carbon-13 and nitrogen-15 are described.

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Theoretical formulas for the prediction of effective thermal diffusivity on one-dimensional unsteady heat conduction of fish and meats in unfrozen and frozen states were investigated. Theoretical results agreed well with measured values in the temperature range of 9∼-22°C. Theoretical results also agreed with published data from Dickerson and Read within the standard deviation of 7.8%. To predict the effective thermal diffusivity of fabricated food materials, a ternary contour diagram was prepared.

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The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris-HCl (20mM, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of chlorophyl-lase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the chlorophyllase activity in the biphasic system decreased from 7.0h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The V_max and K_m as well as the inhibitory effect of phytol on the chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.

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Similar unknown peaks were detected on an amino acid chromatogram of the acid hydrolysates in 3-deoxyglucosone (3DG)-lysozyme as well as glucose-lysozyme reaction systems. Unknown peaks were also detected in the acid hydrolysates of 3DG and N^α-benzoylarginine amide (BzArgNH₂) reaction system. By this system, several products were purified by reversed-phase HPLC and subjected to a FAB-MS analysis. The mass spectral data showed that the major products were formed from one molecule of BzArgNH₂ and from one or two molecules of 3-DG. The major product was identified as 2-(4-benzoylamino-5-pentamide)-amino-5-(2, 3, 4-trihydroxybutyl)-4-imidazolone.

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Two new cercosporins, (+)-isocercosporin (1) and (+)-14-acetylisocercosporin (2), were isolated from culture filtrates of Scolecotrichum graminis Fuckel, which is the causal fungus of a leaf streak disease in orchardgrass. The structure of each was elucidated by spectroscopic analysis, and (+)-isocercosporin (1)is the first isolated enantiomer of known (-)isocercorporin (8b). Compounds 1 and 8b had higher phytotoxic activity than that of (+)-cercosporin (a), an atropisomer of 8b. The results of the bioassay suggest that the phytotoxic activity of cercosporins depends on the relative configuration, and not on the absolute configuration. In addition, three phenol compounds, 1-(2, 4-dihydroxy-6-methylphenyl) ethanone (3), isosclerone (4), and trans-3, 4-dihydro-3, 4, 8-trihydroxy-1(2H)-naphthalenone (5), were isolated as minor components and identified. Compound 3 was isolated for the first time as a natural products.

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A screening was attempted to isolate microorganisms that tolerate high concentrations of H⁺ and ionic aluminum in acidic soils. A new microorganism that can grow in the presence of a high concentration of Al³⁺ at low pH was isolated from acidic tea fields. The microorganism, designated strain ST-3991, has been identified as a Flavobacterium sp. It can tolerate aluminum concentrations up to 2000ppm at pH 3.5. The bacterial growth was affected by the concentration of ionic aluminum. When the bacterium was incubated at an initial pH 3.3 in liquid medium containing 100 ppm aluminum, the pH of the medium increased steeply with time, and the medium reached pH 4.4 after 10 days. The bacterium is considered to be useful to improve acidified soils by decreasing the ionic aluminum concentration, while preserving indigenous terrestrial ecosystems.

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Effects of pH shifted in the stationary phase on the chemical composition of Euglena gracilis cells were examined. When the cells that were cultivated at fixed pH of 4.5 reached stationary phase, the pH of the culture was shifted to 2.0-8.5, and the cultivation was continued for 24 hours more. Shifting pH to below 3.0 and between 6.5 and 7.5 increased cellular protein and decreased paramylon. The maximum content of protein in the cells was 77.5% when the pH was shifted to 7.5. At shifted pH of higher than 8.0, the protein content was greatly decreased and the paramylon content was greatly increased. This technique of shifting pH in the stationary phase did not improve the lipid content or fatty acid composition in Euglena for use as a feed for larval fish. Arachidonic acid, however, was found to be decreased in alkaline pH after shifting. This pH shifting technique that can improve the protein content in Euglena cells is useful for mass production of Euglena for use in the feed industry.

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Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No.9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6-8and at temperatures 50°C or less in the presence of CaCl₂. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased.

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The action of various enzymes on beet pulp was examined to increase the efficiency of enzymatic saccharification. Endogenous enzyme from the homogenate of sugar beet had higher activity for carboxymethyl-cellulose than for beet pulp. The enzyme from formerly isolated strain C had a similar action pattern to that of beet enzyme. The beet homogenate also contained pectin-methylesterase activity, which was clearly measured for the formation of free carboxyl groups in uronic acids by the fluorescent labeling method with water-soluble carbodiimide (EDC). Reduction of uronic acids with EDC-NaBH₄ (Taylor method) stimulated the susceptibility of beet pulp to pectinase and led to the increase in the recovery of neutral sugar, glucose, and galactose. The limit of hydrolysis of beet pulp and EDC-NaBH₄ reduced beet pulp was increased by the combination of pectinase and cellulase (Meicelase). These results gave useful information on the enzymatic conversion of beet pulp for ethanol fermentation by yeast.

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The bioflocculant produced by Rhodococcus erythropolis S-1 was found to exist as huge assemblies, the molecular mass of which is over one million daltons, composed of many polypeptides and lipids in aqueous solution. We have isolated and purified this lipid bioflocculant by ultracentrifugation, extracting with 90% acetone, and two successive silica gel chromatographies from the culture broth. It was homogeneous on silica gel thin-layer chromatography. ¹H-NMR and HPLC studies showed that it was a kind of glycolipid that contained a C₁₆ methylene chain on the average and glucose in its chemical structure. The flocculating activity against kaolin clay suspension was dependent on the Ca²⁺ concentration.

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Application of cryoultramicrotomy to immunoelectron microscopy showed the localization of the chitin synthase of Absidia glauca in situ by using anti-chitin synthase IgG. Mild fixation with a mixture of 0.25% glutaraldehyde and 4% formaldehyde did not destroy the antibody-binding capacity of chitin synthase and preserved the ultrastructure of organelles. Infusion with a mixture of polyvinylpyrrolidone and sucrose as a cryoprotectant before freezing provided adequate sectioning plasticity. Immunolabeling of colloidal gold was detected both at the plasma membrane and the vesicles, and more so in the latter. The results of immunoelectron microscopy were consistent with the fractionation profile of isopycnic sedimentation of the microsomal fraction on a linear sucrose gradient. The profile also demonstrated two peaks of chitin synthase, one of these cosedimented with vanadate-sensitive ATPase and the other with lower buoyant density populations. The former corresponded to the plasma membrane and the later, vesicles in the cytoplasm.

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Cyclodextrin glucanotransferase [1, 4-α-D-glucan 4-α-D-(1, 4-glucano)-transferase, cyclizing ; CGTase, EC 2.4.1.19] from an alkalophilic Bacillus species produced hesperidin monoglucoside and a series of its oligoglucosides by the transglycosylation reaction with hesperidin as an acceptor and soluble starch as a donor. The formation of the glycosides was more effective at alkaline pHs than at neutral or acidic pHs, because of higher solubility of the acceptor. The structure of the purified monoglucoside was identified as 4^G-α-D-glucopyranosyl hesperidin by FAB-MS, α-, β-glucosidase and glucoamylase treatments, and methylation analysis. The solubility of both hesperidin mono and diglucoside in water was about 300 times higher than that of hesperidin, and they were found to have a stabilizing effect on the yellow pigment crocin, from fruits of Gardenia jasminoides, against ultraviolet radiation.

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The active site of isomalto-dextranase from Arthrobacter globiformis was investigated by kinetic and chemical-modification methods. The ionization constants, pK_e1 and pK_e2, of the essential ionizable groups 1 and 2 of the free enzyme were 3.3 and 6.3 for dextran T2000 and 3.5 and 6.1 for isomaltotriose. The PK_e1 and PK_e2 both shifted to higher PH when the dielectric constant of the reaction mixture decreased. The heats of ionization for groups 1 and 2 were 0 kcal/mol or less with both substrates. These kinetic results suggested that the ionizable groups essential for the enzyme activity were carboxyl and carboxylate. Modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, modifying carboxyl residues specifically, resulted in inactivation of the enzyme, and isomaltotriose protected the enzyme against such inactivation. These findings also indicated that the carboxyl groups were essential to the enzyme activity.

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The distribution of α-tocopherol (α-Toc) stereoisomers in the tissues of rats fed on diets containing different levels of all-rac-α-tocopheryl acetate was determined by a newly revised HPLC method and compared with that of RRR-α-Toc fed rats. By this method, all-rac-α-Toc in the blood and tissues was completely separated into 2R-isomers (one peak) and 2S-isomers (three peaks). The concentrations of the 2R-isomers of α-Toc in the blood and tissues of the rats in the all-rac-α-tocopheryl acetate group were significantly higher than those of the 2S-isomers. In most tissues, the level of 2S-isomers was in order of SRS > (SSS+ SSR)/2 > SRR. In the RRR-α-Toc-fed groups, the concentration of RRR-α-Toc in every tissue depended on the dose of α-Toc. When the concentration of 2R-isomers in the tissues of rats fed on all-rac-α-Toc (100 mg/kg diet)is compared with the concentration of RRR-α-Toc in the tissues of rats fed on RRR-α-Toc (50 mg/kg diet)the concentration of 2R-isomers in every tissue of the former is the same as the RRR-α-Toc concentration of the latter.

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A subtilisin inhibitor was purified from the seeds of Canavalia lineata by ammonium sulfate precipitation, ultrafiltration on a YM-30 membrane, column chromatography on DEAE-Toyopearl and SP-Toyopearl, followed by reverse-phase HPLC. The inhibitor (CLSI-I) is a low molecular weight protein (M_r about 6500)containing no half-cystine residue, and quite stable as to extreme heat and pH treatment. CLSI-I inhibited subtilisin-type serine proteases including S. griseus alkaline protease. The amino acids of CLSI-I were sequenced by manual Edman degradation after enzymatic digestion with Achromobacter lyticus lysyl endopeptidase and Staphylococcus aureus V8 protease. CLSI-I contains 65 amino acid residues and showed a high homology to potato inhibitor I family proteins.

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Growth of Escherichia coli K-12 JA300 on agar and in medium in the presence of 10 different organic solvents has been investigated to obtain information on solvent tolerance. Cells grew readily in nutrient medium containing n-octane or isooctane, and some tolerant cells grew in the presence of n-hexane. Cyclohexane and p-xylene were toxic. Bacteriocidal effects of organic solvents agreed with their bacteriostatic effects observed previously. The toxicity correlated with the common logarithm of the partition coefficient of the solvents between n-octanol and water. When 13 cations were assessed for their effects on solvent tolerance, it was found that the organism grown in the presence of Mg²⁺, Ca²⁺, Ba²⁺, or Sr²⁺ had a higher tolerance level towards n-hexane or cyclohexane. The addition of EDTA to medium decreased the solvent tolerance level of the organism.

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The cells of the facultative alkaliphile Bacillus sp. C-125 grown at neutral pH autolyzed rapidly in alkaline buffers of pH 9-10. By contrast, the cells grown at alkaline pH were apparently stable under the same conditions. Alkaline N-acetylmuramyl-L-alanine amidase associated with the cell walls caused this alkali-instability. Meanwhile, cross-linkage between peptide moieties of the peptidoglycan of the organism was dependent on the culture pH. The cross-linking rate was low (31%) in the peptidoglycan of the cells grown at neutral pH, and high (52%) in the cells grown at alkaline pH. This low linking rate is one of the reasons why the cells grown at neutral pH were unstable at alkaline pH. The high linkages in the peptidoglycan might be a cellular adaptation of the organism for growth in alkaline environments.

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Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease ; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5 m and a partially purified protease was obtained. This protease had a molecular weight of more than 400, 000. When the sonicated protozoa were heated at 55°C for 15 min, the active peak from the Bio-Gel A-1.5 m column was shifted to a lower molecular weight, 27, 000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.

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A chromosomal virulence gene, acvB, of Agrobacterium tumefaciens [J. Bacteriol., 175, 3208-3212(1993)] was over-expressed in Escherichia coli. A 47-kDa protein was produced and localized in the periplasmic space of E. coli. Amino acid sequence analysis of its N-terminal demonstrated that a signal peptide of 24 amino acids was cleaved from the pre AcvB protein to produce the mature 47-kDa protein. Western-blot analysis using the antiserum against the AcvB protein detected a 47-kDa protein in the periplasmic space only with strain A208 (acvB⁺). The amount of AcvB protein synthesized was not increased in strain A208 by induction with acetosyringone (100μM). There was observed no significant difference in induction by acetosyringone of virB : : lacZ, virD : : lacZ, and virE : : lacZ fusion genes regardless of the presence or absence of the acvB gene. The T-strand (lower strand of T-DNA) was detected in strains A208 as well as B119 (acvB^-) which were cultured in induction medium containing acetosyringone. AcvB protein bound to single-stranded DNAs with no apparent sequence specificity. The results suggest that AcvB protein binds to the T-strand in periplasm and mediates the transfer of the T-strand from A. tumefaciens to the host plant cell.

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The process of antigen presentation is not well understood. We screened for drugs that distinguish presentation of allogeneic class 2 antigens and exogenous antigens. When spleen cells were used as antigen presenting cells (APC), leupeptin and antipain preferentially inhibited allogeneic class 2 presentation, while they did not affect presentation of exogenous antigen and T cell growth. In contrast, they inhibited both presentations when spleen adherent cells (SAC) were used as APC. Our results suggest that SAC (mainly macrophages) and splenic B cells use different pathways to present exogenous antigens and that pathways to present allogeneic class 2 molecules are similar.

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A new glucuronide saponin (1) was isolated as its methyl ester (2) from the leaves of Camellia sinensis var. sinensis. On the basis of its spectral data and the results of chemical degradation, the structure was elucidated to be 3-O-{β-D-galactopyranosyl(1→2)-[β-D-xylopyranosyl(1→2)-α-L-arabinopyranosyl(1→3)]-β-D-glucuronopyranosyl}-21-O-cinnamoyl-16, 22-di-O-acetylbarringtogenol C.

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A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N, N'-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS-polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0-9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.

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D-myo-Inositol-1-phosphate was synthesized by a short and facile route from optically pure 1D-1-acetoxy-4, 6-di-O-benzyl-myo-inositol, which was easily obtained by a highly regio- and enantioselective enzyme-catalyzed acetylation of 4, 6-di-O-benzyl-myo-inositol.

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The main aroma components of oolong and black tea, linalool and four diastereomers of linalool oxides (LO_s), were enantioselectively isolated by capillary gas chromatography, using a column coated with an optically active liquid phase, permethylated β-cyclodextrin. The R/S ratio varied among linalool and LO_s, and among the different types of tea, the ratio for a particular compound also being different. However, the complete patterns of R/S ratio were similar in the semi-fermented and fermented teas, respectively. Using a specific cultivar of black tea, the R/S ratio for each of the five compounds was compared in the free state in black tea with that of an aglycone of the glycoside in fresh tea leaves or in black tea. While the e. e. values of the compounds varied, those for a specific compound were similar, except for linalool, regardless of their free or combined state. These results show that LO_s are not directly transformed from linalool, but are formed enzymatically from glycoside precursors.

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Various picolinic acid derivatives were synthesized from ammonia and hydroxymuconic semialdehyde derivatives that were oxidatively prepared from various catechols by the action of Pseudomonas catechol 2, 3-dioxygenase (C23O, metapyrocatechase).

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Many isolated strains of Aureobasidium sp. were found to produce extracellular lipids as heavy oils in culture media containing no CaCO₃ as a neutralizing agent. The lipids (35g) were recovered from the culture broth (1 liter) of a light-colored mutant of Aureobasidium sp. A-2 and proved to be a mixture of fatty acid esters of arabitol and mannitol. The two main components of the lipophilic moiety of the lipids proved to be 3, 5-dihydroxydecanoic and 5-hydroxy-2-decenoic acids by the identification of their lactones, (+)-3-hydroxydecan-5-olide and (R)-(-)-2-decen-5-olide, that is, R-(-)-massoilactone, respectively.

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Hypoallergenic wheat products in the form of pasta-like noodles and bread were fabricated. Wheat flour was added with a 0.6-fold weight of water dissolving collagenase to obtain hypoallergenic flour batter. Methods for producing hypoallergenic pasta-like noodles and bread from the batter were designed. In the noodle making, the batter was mixed with an oilgosaccharide with a mild sweetness, a surfactant, and salt, and the mixture was extruded under heating. Both retorting and refrigerating processes were applied to the noodles. In the bread making a mixture of the batter, glucose, citric acid, sodium hydrogen carbonate and salt was baked at 180°C. The addition of glucose contributed to generation of faborable flavor and color.

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A simple and rapid method was developed separating phosphoserine, phosphothreonine, phosphotyrosine, phosphoric acid, and ATP by one-dimensional cellulose thin-layer chromatography, using a freshly prepared solvent system of 1-butanol-formic acid-water (5 : 2 : 1). This system was applied to determine phosphoamino acid (phosphoserine) of wheat elongation factor 1β phosphorylated by casein kinase II from the wheat embryo.

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Three kinds of xylo-oligosaccharides having structures of 3²-β-xylosylxylobiose, 3²-β-xylobiosylxylobiose, and 2²-β-xylobiosyl-xylobiose were isolated from an enzymatic hydrolysate of hard-wood xylan with Streptomyces β-xylanase. The structures suggest that the hardwood xylan has both (1→2)- and (1→3)-β-D-xylopyranosyl linkages in the structure, and the specificity of Streptomyces β-xylanase toward the stubs is similar to that toward glucuronic acid stubs, but is somewhat different from that toward arabinose and xylosylarabinose stubs.

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Homogeneous hydrogenation of 2-phenyl-3-methyl-2-butenoic acid derivatives in the presence of a catalytic amount of such chiral phosphine-rhodium complexes as Rh/(R)-BINAP, Rh/(R)-, or (S)-Cy-BINAP, Rh/(R)-p-tolyl-BINAP, and Rh/(R)-p-methoxy-phenyl-BINAP afforded the corresponding phenylacetic acid de-rivatives in high enantiomeric excesses and in quantitative chemicalyields. The optically active phenylacetic acid derivatives are useful intermediates of agrochemicals and pharmaceuticals.

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A deodorant of methyl mercaptan was isolated from hydrangea (Hydrangea macrophylla Seringe var. otaksa Makino) and determined to be 2, 6-dimethoxy 1, 4-benzoquinone by HR-EI-MS, and ¹³C- and ¹H-NMR.

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Endogenous brassinosteroids in the pollen of Lilium elegans were investigated. Four biogenetically-related brassinosteroids, i. e., teasterone (4), typhasterol (3), castasterone (2), and brassinolide (1), were identified by GC-MS or GC-SIM, suggesting the presence of the biosynthetic pathway, teasterone (4)→typhasterol (3)→ castasterone (2)→brassinolide (1) in the pollen.

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Although fully fermented doughs with a non-freeze-tolerant yeast lost fermentative activity after frozen storage, heat treatment for 46°C for 10 min of the fermented doughs greatly improved the freeze tolerance. The specific volume increased and the proof time decreased. The heat treatment was effective for the straight method of white dough and also for the sponge and dough methods of sweet dough.

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We purified and studied two L-stereoselective carbonyl reductases from bakers' yeast (Saccharomyces cerevisiae). One catalyzed exclusively the enantioselective reduction of carbonyl compounds such as β-keto esters and the other acted on α-acetoxy ketones and β-keto esters. The enzymes had identical molecular weights and catalyzed the L-stereoselective reduction of various carbonyl compounds with similar substrate specificity, but they were different proteins coded by different genes.

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A mutant strain, G7, of Acetobacter sp. BPR2001 incapable of producing pyrroloquinoline quinone (PQQ) was obtained by chemical mutagenesis. The strain was defective in the activities of membrane-bound glucose dehydrogenase and alcohol dehydrogenase, both of which known to contain PQQ as their prosthetic group. However, the mutant had almost the same level of activity as the parental strain of membrane-bound aldehyde dehydrogenase, which also has been suggested to be a quinoprotein with PQQ. Our findings showed that PQQ was not the prosthetic group of membrane-bound aldehyde dehydrogenase.

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A Lactobacillus plantarum strain, LTF154, isolated from a fermented sausage, produces a bacteriocin, designated plantacin 154. Plantacin 154 was stable to heat treatment, and its activity was sensitive to proteolytic enzymes. The molecular mass, as indicated by activity detection after SDS-PAGE, was estimated to be 3.0 kDa or less. A plasmid-curing experiment and transformation analysis indicated that a 9.5-MDa plasmid, pLP1542, may be involved in the production of plantacin 154.

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A Mg²⁺-ATPase was solubilized from membranes of Acetabular-ia cliftonii using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. One active ATPase fraction after Mono Q chromatography had a specific activity of 10 units/mg of protein. Judged from subunit composition [54 (a), 50 (b) with a fainter band around 40kDa], catalytic properties, and N-terminal amino acid sequence of the b subunit, the isolated enzyme was comparable to the Cl^- -ATPase of Acetabularia acetabulum. Immunological characterization of both subunits showed significant similarity to the F type of ATPase. Cl^- -transport activity was observed by reconstitution studies into liposomes.

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Five alkali-sensitive mutants of facultative alkaliphilic Bacillus C-125, which maintain low internal pH, showed cellular morphological abnormalities, elongation, and curling. It is considered that alkaliphiles adapt to alkaline environments primarily by a membrane pH homeostasis mechanism ; while cell-surface functions can contribute to their alkali-tolerance.

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A fungus, Paecilomyces canadensis NC-1, that decolorizes a coffee solution was isolated by enrichment culture. This fungus decolorized a coffee solution by 79% under the optimal conditions. The larger molecular weight fractions of coffee pigments were adsorbed to the mycelia, and the autoclaved mycelia also adsorbed coffee pigments. This fungus decolorized such phenol-type pigments as those found in coffee and black tea extracts.

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By use of the promoter probe transposon Tn5-B21, two promoter fragments were isolated. One promoter (822 bp) (GC = 63. 5%)isolated from P. putida loses all of its promoter activity by exo-nuclease or restriction enzyme deletion. Another promoter (264bp)(GC = 49.2%) isolated from P. fluorescens could be shortened to 154 bp by exonuclease deletion without any effect on its promoter activity in several Pseudomonas species.

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The proline permease gene of Pseudomonas fluorescens has been isolated from the promoter region isolated by using a promoter probe (transposon Tn5-B21). By DNA sequencing of 2222 bp the primary structure of permease (494 aa) was deduced. The DNA sequence is 71. 0% and 70. 7% identical and the amino acid sequence is 75. 8% and 76. 0% similar to those of Escherichia coli and Salmonella typhimurium, respectively.

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We detected carboxymethyl cellulase activity in a crude extract of Acetobacter xylinum KU-1. The enzyme activity was detected when glycerol, D-fructose, D-mannitol, D-glucose, D-arabitol, D-sorbitol, or carboxymethyl cellulose was used as a carbon source. The optimum pH was found to be 4.0, while the optimum temperature was 50°C. The enzyme activity was inhibited characteristically by the addition of Hg²⁺.

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Sequential deletions of the promoter region of the WCI-3b gene, which encodes the major chymotrypsin inhibitor of winged bean, were constructed and their expression was analyzed in transgenic tobacco plants and in bombarded winged bean seeds. In transgenic tobacco plants, a critical promoter region which is important for high levels of expression in seeds was identified, but deletion of this region had essentially no effect when bombarded into winged bean seeds.

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An aminopeptidase P, capable of hydrolyzing oligoproline, was isolated from the homogenate of bovine brain. The molecular mass of the enzyme was 140kDa by gel filtration. The enzyme was activated by Mn²⁺ and inhibited by o-phenanthroline. The enzyme hydrolyzed substrates such as Pro-Pro-Pro-Pro, Pro-Pro-Pro, and Pro-Pro to proline, and cleaved N-terminal amino acids from pep-tides containing penultimate prolines such as bradykinin and neuro-peptide Y.

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Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.

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Gram-positive bacteria, identified as Rhodococcus erythropolis, were isolated from the ecosystem of the wood-feeding termite Reticulitermes speratus and found to aerobically degrade poly-chlorinated biphenyl (PCB) compounds. Rhodococcus erythropolis strain TA421 and strain TA431 were isolated by enrichment culture from termites obtained from different locations and each was found to be capable of degrading polychlorinated biphenyl (PCB)compounds to chlorobenzoates. These results suggest that the termite ecosystem is one possible habitat for biphenyl- and PCB-degrading Rhodococci. The spectrum of PCB-congeners degraded by strain TA421 is different from that of other, previously characterized PCB-degrading bacteria such as Rhodococcus globerulus strain P6(formerly Corynebacterium sp. strain MB1) or Pseudomonas sp. strain LB400.

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Since sesamin influences the metabolism of essential fatty acids, its effects on cholesterol metabolism and on the incorporation of linoleic acid were studied by using cultured rat artery smooth muscle cells (SMCs) and primary cultured rat hepatocytes. Cholesterol synthesis from acetate was inhibited by sesamin in SMCs, and the distribution of incorporated linoleic acid in the lipid and phospholipid subfractions was altered by sesamin in rat hepatocytes.

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Effects of L-tryptophan and its metabolites were evaluated on synthesis of nerve growth factor (NGF) in cultured mouse astroglial cells. L-Tryptophan stimulated NGF production in a dose-dependent fashion. Serotonin and quinolinic acid slightly increased NGF synthesis. L-Kynurenine had a marked stimulatory effect on NGF synthesis at a dose of 100μM. In contrast, kynurenic acid had no effect.

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Histamine oxidase (EC class 1.4.3) was found in cells of Arthrobacter globiformis IFO 12137 (ATCC 8010) grown on histamine. The enzyme purified to a specific activity of 9.4 units/mg had a purity of at least 80%. The enzyme oxidized histamine with concomitant formation of H₂O₂. Phenylethylamine, dopamine, aromatic monoamines, and aliphatic mono- and diamines were oxidized at lower rates. Cu²⁺ -chelators inhibited the enzyme, indicating that the enzyme is Cu²⁺ -dependent. Carbonyl-blocking reagents also inhibited the enzyme. The enzyme catalyzed the Nitro Blue Tetrazolium/glycinate reaction, which is characteristically catalyzed by quinones and quinoproteins. These results strongly suggest that the enzyme contains a quinonoid cofactor.

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Unlike Borgstrom's theory that pancreatic colipase overcame the inhibition of pancreatic lipase activity caused by bile salts in tributyrin, pancreatic lipase activity in triolein was activated by bile salts and synergistically accelerated by colipase in the presence of NaCl and CaCl₂. This finding reflected more closely the behavior of pancreatic lipase by using triolein as a representative dietary fat.

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