The nature of the binding of saccharides to arbin-b, a toxic lectin isolated from Abrus precatorius seeds, was studied by equilibrium dialysis and fluorescence spectroscopy. Equilibrium dialysis data indicate that abrin-b has two saccharide-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site), to which both galactopyranosides and N-acetylgalactosamine can bind. With excitation at 290 nm, abrin-b displayed a fluorescence spectrum with an emission maximum at 345nm. Upon binding with specific saccharides, this spectrum shifted to a wavelength shorter by 5 nm, suggesting that saccharides bind to abrin-b in such a manner as to induce a change in the environment of the tryptophan residue or residues at or near the respective binding sites. From the variation of fluorescence at 320 nm with saccharide concentrations, the association constants for binding of saccharides to the respective sites were measured. The results suggest that the HA-site has a subsite favorable for saccharides having β-1, 4 linked galactopyranoside at the non-reducing end like lactose in addition to the galactose-recognition site, while the LA-site may not have such a subsite.

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In order to increase the yield of ML-236B, an intermediate for pravastatin Na (an inhibitor of cholesterol synthesis) production, the improvements of an ML-236B producing strain of Penicillium citrinum, the medium composition, and the culture conditions were studied. A mutant strain S-5808, which produces ML-236B 20 times more than the original strain does in flask culture, was isolated. As the cells of S-5808 required a large amount of the carbon source substrate to produce a lot of ML-236B, a fed-batch culture method was developed. The continuous feeding of carbon source (glycerol or mixture of glycerol and maltose)was very effective for the increase of ML-236B production. ML-236B in the culture broth became oily with lowering of pH during cultivation, adhered to the cell surface, decreased the carbon source consumption and finally stopped the ML-236B production at a late period of fermentation. The addition of a polypropylene glycol type surfactant enhanced the ML-236B productivity. The addition of the surfactant released oily ML-236B from the cell surface, therefore oxygen uptake, carbon source consumption, and ML-236B production continued throughout the fermentation.

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Copper metallothioneins were extracted from Aspergillus niger after an induction period of five hours in a copper-containing suspension medium. The crude extract (FI) was obtained by cell homogenization and the partially purified extract (FII) was obtained by heat treatment at 60°C for 10 min. The partially purified extract (FII) was further purified by successive chromatographies on size-exclusion (Biogel P-30) and ion-exchange (TSK-HW 65-DEAE) chromatography columns. All protein fractions were analyzed for their protein and induced copper contents. The Cu/protein ratio increased steadily throughout the purification process ; the most purified fraction (FIV) showed a 24-fold increase, with a recovery of 13%. The molecular weight of the Cu-metallothionein fraction FIV was 11, 000 and the ratio of moles of copper per mole of protein was 6.6.

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The effects of polyamines on the ice-nucleating activity of an ice-nucleating bacterium, Erwinia uredovora KUIN-3, were investigated. The activity in the outer membrane derived from this bacterium was increased by the addition of spermidine (10mM). Also, the activity was stabilized in alkaline pH buffer with the addition of polyamine. Further, the addition of methylglyoxal bis(guanylhydrazone) which inhibited the activity of S-adenosylmethionine decarboxylase, inhibited the ice-nucleating activity of the cells of this bacterium. The amount of polyamine in this bacterium was examined using HPLC. The components of the polyamines were three species, putrescine, cadaverine, and spermidine. The amounts of polyamines in the cells and outer membranes after MGBG treatment decreased. The results suggested that polyamine was the most important component of the ice-nucleating material in this bacterium.

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The extracellular ice-nucleating matter (EIM) produced by the ice-nucleating bacterium, Erwinia uredovora KUIN-3, was purified to apparent homogeneity by ultrafiltration, sucrose density-gradient ultracentrifugation, and gel filtration. The purified EIM was sphercial matter (0.2-0.4 μm) by examination using a transmission electron microscope. The ice-nucleating activity from the EIM was equal to that of the cells in this strain. It had become apparent that the EIM had class A (∼ -5°C), B (-5 ∼ -8°C), and C (-8°C ∼) structures as judged by its freezing difference spectrum in D₂O versus H₂O. The enzyme treatments by protease K, α-mannosidase, β-galactosidase, and phospholipase C decreased the activity of the EIM as well as of the cells. Further, the activities of class A and B from the EIM were decreased by treatment with diamine oxidase. Also, the activity of the purified. EIM was stable from pH 5.0 to 10.0 and at temperatures below 25°C. It was demonstrated that the components of the EIM were lipid (10%), protein (43%), saccharide (35%), and polyamine (12%).

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Several 4'-C-methylnucleosides were prepared. ¹H-NMR studies on these nucleosides showed that they have the 3'-exo furanose ring conformation different from the 3'-endo conformation of natural nucleosides.

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The present study is the first to report the hypocholesterolemic effect of chitosan on humans. When 3-6 g/day of chitosan was given in the diet to 8 healthy males, total serum cholesterol significantly decreased, and when the ingestion was stopped, the value increased to the level before ingestion. Serum HDL-cholesterol was increased significantly by the ingestion of chitosan. The excreted amounts of primary bile acids, cholic acid and chenodeoxycholic acid, into the feces was significantly increased by the ingestion of chitosan, and the amount of cholic acid excretion decreased significantly after the ingestion was stopped. These facts suggest that chitosan combined bile acids in the digestive tract, and that the combined product was excreted into the feces. This, in turn, deceased the resorption of bile acids, so that the cholesterol poool in the body was decreased and the level of serum chrolesterol consequently decreased.

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Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several μm. The encapsulation effiiciency was improved by increasing the rehydration temperature to 60°C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.

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The structure of dextran synthesized from maltotetraose by dextrin dextranase (EC 2.4.1.2) from Acetobacter capsulatus ATCC 11894 was analyzed. When the Acetobacter dextran (AD) was acetolyzed, glucose and maltose were produced. AD was allowed to react with α-amylases. AD was digested by bacterial saccharifying α-amylase and bacterial liquefying α-amylase, and glucose, maltose, and maltotriose were produced. The structure of the fraction obtained from dextranase-digested AD by activated charcoal chromatography, which did not contain glucose, isomaltose, and isomaltotriose, was investigated by methylation analysis, and the ratio of 2, 3, 4, 6-tetra-O-methyl- : 2, 3, 4-tri-O-methyl- : 2, 3, 6-tri-O-methyl- : 2, 3-di-O-methyl-alditol acetate was estimated as 22.9 : 46.8 : 15.5 : 14.8. This result indicated the existence of α-1, 4 branches and that of α-1, 4 linkages in α-1, 6 glucosyl linear chains. Native AD was calculated to be constructed with 6.23 branching points and 6.53 α-1, 4 linked glucosyl residues per 100 glucosyl units. Though AD was digested slightly by rat intestinal acetone powder, high molecular weight polymers remained. Therefore AD could be used as a dietary fiber.

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The nucleotides of the Mn-superoxide dismutase (SOD) gene of Bacillus circulans and the Fe-SOD gene of Aerobacter aerogenes were sequenced by PCR. These SOD genes were specifically amplified by using oligonucleotide primers corresponding to the amino-terminal amino acid sequences and the antisense strand primer corresponding to the common amino acid sequence near the carboxyl-terminus among various Mn- and Fe-SODs thus far sequenced. The PCR products amplified from B. circulans and A. aerogenes genes contained a 486-nucleotide sequence encoding 162 amino acids and a 507-nucleotide sequence encoding 169 amino acids, respectively. Each sequence seemed to contain most of the open reading frame encoding the SOD protein when compared with other sequenced SODs. The two amino acid sequences deduced from the nucleotide sequences of PCR products had an identity of 66.1%. However, the SODs from B. circulans and A . aerogenes were immunologically distinct from each other judging from an immunoprecipitation test. The two SODs had high homologies with other bacterial Mn-SODs, especially the highest homology of 75. 4% and 66.7%, respectively, with the B. stearothermophilus Mn-SOD. Genomic Southern hybridization suggested that each PCR product of the bacterial genes that were synthesized and sequenced was the product of the sole SOD gene in each bacterium.

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The plasma cholesterol-lowering effect of dietary glycine was investigated in relation to its influence on the free amino acid profiles in the liver and plasma and on the composition of phospholipids in liver microsomes of rats fed on a cholesterol-free diet. The plasma total and HDL cholesterol levels were significantly decreased by dietary supplementation with glycine and ethanolamine, but not with serine and threonine. The hepatic free ethanolamine concentration was enhanced by dietary supplementation with glycine as well as with ethanolamine, and there existed a significant Correlation between the hepatic free ethanolamine concentration and plasma total or HDL cholesterol level. Dietary supplementation with glycine significantly increased the content and proportion of phosphatidylethanolamine in liver microsomes and inversely decreased the proportion of phosphatidylcholine. The phospholipid composition of liver microsomes changed prior to the plasma total cholesterol level in response to glycine supplementation, suggesting that the alteration of phospholipid composition was not the result of an alteration of the plasma cholesterol level. From these results, it is suggested that the plasma cholesterol-lowering effect of dietary glycine might be associated with the alteration of microsomal phospholipid composition.

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In order to search for a chitosan with low crystallinity ; partially N-deacetylated chitins (PDC) and partially N-acetylated chitosans (PAC) with a low degree of N-acetylation (DAc) were examined by X-ray powder diffraction measurements. Three PDC samples, having less than 30% DAc and prepared by solid-state deacetylation, gave X-ray powder patterns showing the presence of α-chitin, a hydrated crystal of Chitosan, or their mixture, respectively. When these PDC samples were treated with an acid-alkali, however ; reduced crystallinity was observed. By annealing in water at 160 or 200°C, the latter PDC having DAc less than approx. 22% gave powder patterns indicating the presence of an anhydrous crystal which may spoil the chitosan's functionality. In contrast, PAC prepared by N-acetylating pure chitosan (DAc = 0%) in a swollen state, which can be expected to have random copolymers in the chain, was always less crystallized than PDC, this crystallinity depending on the molecular weight. In the case of high-molecular-weight PAC samples, whose DAc was in the range of 5-21%, the effect of high molecular weight on reducing crystallinity was larger than that of the degree of N-acetylation.

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Cathepsins L and L-like (58kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30, 000 and 58, 000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (K_D) of cathepsins L and L-like proteinases at 50°C were 5.1 × 10^-5 and 6.9×10^-4s^-1, respectively. K⁺, Na⁺, Mg⁺, and Sr⁺ did not affect them, while Zn²⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Fe²⁺, and Fe³⁺ inhibited the activity of the purified catepsins L and L-like proteinase.

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Nicotiana tabacum was transformed with a chemically synthesized gene encoding the human epidermal growth factor (hEGF) under control of the CaMV-35S promoter. The hEGF gene sequence was present at one to several copies in the primary transformant plants (R0), and a transcript with the expected length was produced. Slot blot analysis of total RNAs of the progeny (R1) seedlings, originating from self-pollination of the R0 plants, showed that the level of mRNA expression was generally, but not always, heritable. The highest hEGF peptide content per unit of total soluble protein in young (upper) R1 leaves so far examined by an immunological method was about 0.001%. These results suggest that either the hEGF peptide was less stable than the average leaf protein, or the hEGF mRNAs were not efficiently translated.

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Phthalate oxygenase was induced in Rhodococcus erythropolis S-1, a Gram-positive bacterium, when this bacterium was cultured in a medium containing phthalate as a sole carbon source. The enzyme was purified 118-fold with 4.7% activity yield. The purified enzyme appeared homogenous on native PAGE. This enzyme is a large protein (213 kDa), a tetramer of identical 56 kDa monomers, and a flavoprotein Containing FAD with NADH-dependent dioxygenase activity. The enzyme is specific for phthalate and other closely related aromatic compounds. Optimum pH and temperature were 6.5 and 40°C. The K_m for phthalate and NADH were 0.040 mM and 0.069 mM. The enzyme catalyzes dihydroxylation of phthalate to form 3, 4-dihydro-3, 4-dihydroxyphthalate with consumption of NADH and oxygen.

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Three types of monohydroxybenzoate oxygenase, salicylate 5-oxygenase (SAL5O) forming gentisate from salicylate, m-hydroxybenzoate 6-oxygenase (MHB6O) forming gentisate from m-hydroxybenzoate, and p-hydroxybenzoate 3-oxygenase (PHB3O) forming protocatechuate from p-hydroxybenzoate, were purified from a cell-free extract of Rhodococcus erythropolis S-1, a Gram-positive bacterium. Each purified enzyme was homogenous on native PAGE. Each enzyme was a tetramer having identical subunits, a flavoporotein containing FAD, and a NADH-dependent monooxygenase. The three enzymes were much alike in general enzymatic properties, but very different in substrate specificity.

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Four new polyacetylenic glucosides isolated from the leaf of Jerusalem artichoke (Helianthus tuberosus L. ) were characterized as methyl β-D-glucopyranosyl helianthenate C (5), D (6), E (7), and F (8). The absolute stereochemistry of the glucosyloxymethine was also determined to be of R configuration by preparing the relative compounds with Sharpless asymmetric epoxidation as the key step and source of optical activity.

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Enzymatic production of D-Glu was investigated by the succesive reactions of a glutamate racemase (EC 5.1.1.3) and a glutamate decarboxylase (EC 4.1.1.15) on L-Glu. Lactobacillus brevis ATCC8287 was chosen as a source of glutamate racemase. This strain produced a glutamate decarboxylase simulataneously. The glutamate racemase activity in the cell free extracts was 0.035 units/mg protein. The enzyme kept its activity even at 500 mM of L-Glu (74 g/liter). The optimum pHs of the racemase and the decarboxylase were at around 8.5 and below 4.0, respectively. Both enzymes had no activity at the optimum pH for the other enzyme. L-Glu was racemized first by the glutamate racemase at pH 8.5, then the pH was shifted to 4.0 at which L-Glu was decarboxylated by the glutamate decarboxylase. Starting from 100 g/liter of L-Glu, 50 g/liter of D-Glu was produced and no L-Glu remained in the reaction mixture.

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The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kutzing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens's plasmid, pFTB14, B. subtilis BAA1's pBAA1, and B. coagulans's pBC1, respectively, when conservative amino acid substitutions were included. Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates. The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803. Novel features of pMA1 are discussed.

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The substrate specificity of honeybee α-glucosidase II, a monomeric protein, was investigated in de-tail. The enzyme hydrolyzed phenyl α-glucoside and p-nitrophenyl α-glucoside more rapidly than maltooligosaccharides. Unusual kinetics were observed in hydrolysis of sucrose, turanose, kojibiose, and soluble starch. The s versus v plots showed sigmoidal curves, and so the Lineweaver-Burk plots became concave, indicating positive kinetic cooperativity. The Hill coefficients for sucrose, turanose, kojibiose, and soluble starch were calculated to be 1.46 ; 1.34, 1.15, and 1.28, respectively. The K_m values for these substrates were calculated as the concentration at one half of the maximum velocity. The ratios of the maximum velocities for maltose, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose, maltodextrin (DP=13), kojibiose, nigerose, isomaltose, sucrose, turanose, phenyl α-glucoside, p-nitrophenyl α-glucoside, phenyl α-maltoside, and soluble starch were estimated to be 100 : 163 : 159 : 156 : 149 : 144 : 113 : 96.9 : 92.2 : 31.8 : 81.5 : 68.8 : 268 : 341 : 127 : 17.0, and the K_m values for these substrates, 5.4, 4.0, 6.3, 11, 31, 50, 50, 7.6, 20, 5.6, 6.7, 7.7, 1.6, 1.8, 2.0, and 9.4 mM, respectively. The enzyme was also active on α-glucose-1-phosphate. Its maximum activity was 79.9% of that to maltose and the K_m was 50mM. The substrate specificity of this enzyme was different from that of honeybee α-glucosidase I. The two enzymes were found to be immunologically distinct proteins. Based on the rate parameters for maltooligosaccharides, the subsite affinities (A_i's) in the active site of the enzyme were evaluated. Subsites 1, 2, and 3 having the positive A_i value (A₁, A₂, and A₃ : 0.672, 4.61, and 0.483kcal/mol, respectively) were considered to be effective for binding of substrate to the active site.

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The stereochemistry of 1-methyl hydrogen 3-phenyl-2, 2'-iminodipropionate (BSF-A, 1) was determined to be 2S, 2'S by comparing the spectral data of 1 with those of synthetic derivatives. The effects of BSF-A (1) and its analogs on the growth rate of lettuce seedlings were examined. Both diastereomers, 3a (2S, 2'S)and 3b (2S, 2'R), of dimethyl 3-phenyl-2, 2'-iminodipropionate and a regioisomer, 1'-methyl hydrogen 3-phenyl-2, 2'-iminodipropionate (2), exhibited biological activity similar to that of natural product BSF-A (1).

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The nucleotides of a gene for the extracellular 87-kDa β-1, 3-glucanase of Bacillus circulans IAM1165 and its flanking regions were sequenced. The sequence showed an open reading frame for 877 amino acids, which corresponds to a precursor of the β-1, 3-glucanase. The coding region of 2631 bp is flanked by putative promoter and transcription terminator sequences. The signal peptide was considered to be consisted of 38 amino acids. The amino acid sequence of the mature enzyme composed of 839 amino acids showed high homology to that of the enzyme from B. circulans WL-12, although these enzymes are different in their sizes. A catalytic domain of the enzyme was estimated central region of the sequence on the basis of comparison of amono acid sequences of β- 1, 3- or β- 1, 3 : 1, 4-glucanases. Properties of the periplasmic enzyme produced in Escherichia coli carrying the gene were identical with those of the extracellular enzyme produced by B. circulans IAM1165.

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2-Deoxy-2- [(2R, 3S) -2-fluoro- 3-hydroxytetradecanamido ]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S, 3R)-isomer were respectively synthesized from allyl 2-[(2R, 3S)-3-(benzyloxycarbonyloxy)-2-fluorotetradecanamido]-2-deoxy-4, 6-O-isopropylidene-β-D-glucopyranoside and its corresponding (2S, 3R)-isomer. Both target compounds did not activate macrophage, but the (2S, 3R)-analogue strongly inhibited the binding of LPS to macrophage.

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The atpA gene of pea mitochondria coding for the α-subunit of F₁F₀-ATPase (ATP synthase) is carried on four types of genomic segments generated by recombination, and two of these segments contain the atp9 gene coding for the subunit 9 of F₁F₀ATPase in the 5'-upstream region of atpA in an opposite orientation. The atpA and atp9 genes were separated by a spacer of 2112 bp, which does not contain any open reading frame. Pea mitochondria contained at least four atpA transcripts of 4.5, 2.7, 2.3, and 1.8 kb and at least three atp9 transcripts of 1.35, 0.6, and 0.4 kb. Mapping of these transcripts by northern blot hybridization, primer extension, and S1-nuclease mapping indicated that multiple transcripts of atpA differ in their length both at their 5'- and 3'-termini. On the other hand, three transcripts of atp9 differed in their length only at their 5'-termini. It was found that the 5'-terminal sequence of the 4.5-kb transcript of atpA is complementary to the 5'-terminal part of the 1.35- and 0.6-kb transcripts of atp9 for 705 and 4 nucleotides, respectively.

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To examine the proteolytic activities of various truncated derivatives of the potato virus Y (PVY)50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.

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We have been attempting to confirm the hypothesis that the excretion ratio of nicotinamide metabolites, [N¹-methyl-2-pyridone-5-carboxamide (2-Py) + N¹-methyl-4-pyridone-3-carboxamide (4-Py)]/N¹-methyl-nicotinamide (MNA), reflects the adequacy of amino acid nutrition, but not of niacin nutrition. It is known that methionine and threonine supplementation to a protein-free diet reduced body weight loss. In this paper, we investigated whether rats fed with a protein free-diet supplemented with methionine and threonine would result in this excretion ratio being increased or not. The body weight loss was markedly reduced by the supplementation with both amino acids, as has been reported, and under the conditions, the excretion ratio significantly increased. The activity of 4-Py-forming MNA oxidase, which controls the change in excretion ratio, also increased significantly. From the present results and our previous results, it was proved that the excretion ratio of nicotinamide metabolites, (2-Py + 4-Py)/MNA, reflects the adequacy of amino acid nutrition, but not of niacin

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Maleate hydratase, which hydrates maleate to form D-malate, was purified from a crude extract of Arthrobacter sp. strain MCI2612 by DEAE-Toyopearl, Octyl-Sepharose CL-4B, and Ether-5PW column chromatographies. The enzyme was activated by sulfhydryl compounds and ferrous ion. The overall purification was 44.3-fold with a yield of 3.4%. The molecular weight of the enzyme was 90, 000 by TSK G3000 SW column chromatography. The maleate hydratase appeared as two bands corresponding to molecular weights of about 58, 000 and 28, 000 on SDS-polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 8.5 and 45°C, and was inactivated by chemical agents such as hydroxylamine, p-chloromercuribenzoate, o-phenanthroline, and 2, 2'-dipyridyl. The K_m for maleate was 3.85mM.

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Controlled heating in a dry state greatly improved the surface functional properties of whey proteins (β-lactoglobulin and α-lactalbumin). Although whey proteins were completely insolubilized by heating at 80°C in an aqueous solution, their solubility was kept even after heating at 80°C in a dry state (7.5% moisture content) for 5 days. The surface hydrophobicity of α-lactalbumin was increased during the dry-heating, while that of β-lactoglobulin was decreased. In addition, the fluorescence spectra excited at 280 nm of dry-heated whey proteins suggested the significant conformational changes. High-performance gel chromatography showed that a considerable amount of soluble aggregates was formed in the dry-heated β-lactoglobulin, while a small amount of soluble aggregate was observed in the dry-heated α-lactalbumin. The foaming properties of dry-heated whey proteins were increased to about 3 times that of untreated proteins. The emulsifying properties of dry-heated whey proteins were also increased, compared to untreated proteins, although a slight decrease in the emulsion stability was observed in dry-heated β-lactoglobulin. The improvement of the surface properties seemed to come from the partial unfolding suitable for the formation of foam film and the entrapment of oil droplets.

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(3S, 4R, 6Z, 9Z)-3, 4-Epoxy-6, 9-heptadecadiene (1a) and its antipode (1b) were synthesized by employing the Sharpless asymmetric epoxidation as the key reaction.

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In order to elucidate the interaction of lactic acid bacteria with the immune system, immune responses to the lactic acid bacteria, Bifidobacterium longum and Lactobacillus acidophilus, were examined in mice fed with each organism. In mice fed with B. longum for more than 8 weeks, an antibody response was detected to the cytoplasm of B. longum, but not to the cell wall. On the other hand, in mice fed with L. acidophilus for more than 6 weeks, an antibody response was detected to both the cytoplasm and cell wall of L. acidophilus. Moreover, feeding each organism for 2 weeks enhanced the proliferative response of Peyer's patch (PP) cells to the cell fraction against which the serum antibody was detected. However, this was not found with spleen cells. These results suggest that mucosal stimulation by lactic acid bacteria may induce a systemic immune response to them.

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Dimethoate, an organophosphorus insecticide, inhibited the acetylcholinesterase of the brain of a common carp, Cyprinus carpio, by increasing the K_m without affecting the V_max. A Dixon's Plot confirmed the competitive nature of the inhibition, yielding a K_i of 2×10^-3M. The assay of brain acetylcholinesterase is thus useful in assessing pesticide toxicity to fishes.

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Geraniin, an antimicrobial hydrolyzable tannin against fish pathogenic bacteria, was isolated from Nymphaea tetragona (Nymphaeaceae).

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In a search for nematocidal compounds from wild plant sources, chavicol and demethyleugenol were found from V. furcatum, and 4-vinylphenol was found from three Rosaceae species. These three compounds each contained both p-hydroxyphenyl and exo-methylene moieties, and several related compounds were prepared and tested for nematocidal activity. The results show that the hydroxyphenyl group was essential for the activity, while the aliphatic substituents on the benzene ring enhanced it. The strongest activity was found with 4-propenylphenol.

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The positional isomers of hydroperoxide prepared from methyl linoleate and methyl linolenate by photosensitized oxidation were analyzed by using the hydroperoxide-specific system of chemilumi-nescence-HPLC. Each isomer separated by HPLC was collected, reduced and identified by a mass spectral analysis as the trimethylsilyl derivative. Resolution of a racemate of the positional isomer established by chiral-phase HPLC.

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Several nitrogen bases such as benzo-[h]-quinoline, with no chelating ability, were potent inhibitors of cyanide-resistant respiration of mitochondria isolated from Hansenula anomala. These compounds appear to be a new class of inhibitors of cyanide-resistant respiration, in addition to iron chelators such as 8-hydroxyquinoline and hydroxamates, radical scavengers, and ionophores.

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We detected dye-linked D-mannitol dehydrogenase activity in the crude extract of Acetobacter xylinum KU-1. The enzyme activity was specific for D-mannitol, and not pyridine nucleotide (NAD⁺, NADP⁺)-dependent. The optimal pH was found to be 5.0, while the optimal temperature was at 50°C. The enzyme activity was inhibited by p-quinone noncompetitively.

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The linA gene from Pseudomonas paucimobilis was highly expressed in Escherichia coli, and the linA product (LinA), named γ-HCH dehydrochlorinase, was purified to homogeneity. LinA released three chloride ions per one molecule of γ-HCH. Degradation assay of halogenated compounds by purified LinA showed that the substrate specificity of LinA is very narrow.

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A new compound named BSF-A (1) as a plant growth regulator was isolated from the culture filtrate of Botrytis squamosa, and the structure of 1 was elucidated to be 1-methyl hydrogen 3-phenyl-2, 2'-iminodipropionate by the spectroscopic data of 1 and its methyl ester (2). At a concentration of 300ppm, 1 and 2 promoted root growth by about 220% compared to that of the control.

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Changes in the kind and level of endogenous gibberellins (GAs) in the developing liquid endosperm of tea (Camellia sinensis L.) were investigated. Gibberellin A₁ (GA₁), GA₈, GA₁₉, GA₂₀, and GA₄₄ were identified by GC-MS or GC-SIM. Besides these early C-13 hydroxylated GAs, GA₃, iso-GA₃, and GA₃₈ were also identified. Of these GAs, GA₁, and GA₃ were the major gibberellins. The levels of these GAs were at a maximum in the globular embryo stage and then decreased rapidly during embryo maturation.

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Within a microbial screening two bacterial strains were identified that epoxidize carbamazepine (CBZ) to 10, 11-epoxy-CBZ. Ten strains could hydroxylate 10, 11-dihydro-CBZ to 10, 11-dihydro-10-hydroxy-CBZ. All active organisms belonged to the genus Strep-tomyces. Both reactions were done in a preparative scale using S. violascens ATCC 31560. The culture conditions for the hydroxylation with this strain were improved.

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A xyloglucan was isolated from the 24% KOH extract of gobo (edible burdock, Arctium lappa L.). A methylation analysis and enzymic degradation studies on the polysaccharide showed that gobo-xyloglucan was built up predominantly of repeating-oligo-saccharide units of hepta-(Glc : Xyl=4 : 3), nona- (Glc : Xyl : Gal : Fuc=4 : 3 : 1 : 1) and deca- (Glc : Xyl : Gal : Fuc=4 : 3 : 2 : 1) saccharides in an approximate molar ratio of 14 : 12 : 5, which are the typical structural units of dicot xyloglucans.

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An enzymatic method for assaying methylmalonyl-CoA mutase was devised. The method employs a succinyl-CoA transferase and β-hydroxyacyl-CoA dehydrogenase-coupled reaction to make the formation of NAD⁺ stoichiometric with the amount of the succinyl-CoA formed by the mutase. This method is one of the most convenient and useful assays for the activity of methylmalonyl-CoA mutase in biological samples.

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A bacterium that was isolated from soil was found to produce (S)-(-)-1-phenyl-1, 3-propanediol (SPPD) from the racemate and was identified as a Serratia sp. Serratia sp. No. 2664 produced SPPD with > 99% e. e.

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Protoplasts from four alkaliphilic strains of Bacillus spp. regenerated their cell walls and grew on a hypertonic medium. The regeneration was effective at neutral pH but not at alkaline pH. The protoplasts seemed not to be tolerant of the alkaline pH that is optimum for growth of the strains.

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A cytochrome P450 was purified from microsomes of Rhodotorula minuta. The optical spectrum of the purified cytochrome was characteristic of a low-spin ferric heme protein. lsovalerate caused a type I spectral change in it. The amino-terminal sequence of the cytochrome was different from those of other known microsomal cytochrome P450s. These results indicate that the cytochrome, which is tentatively named P450rm, is a novel species of cytochrome P450.

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FK-565 restored the number of granulocytes in the bone marrow in mice treated with mitomycin C (MMC). Granulocyte counts recovered to the normal level 3 days after MMC treatment at a dose of 0.1mg/kg of FK-565. The restorative activity of FK-565 was more effective than that of muramyldipeptide (MDP) or lipopolysaccharide (LPS).

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Strong fibrinolytic enzyme was purified from the lysate of Katsuwonus pelamis digestive tract (Japanese traditional fermented food, "shiokara"). The enzyme was an alkaline trypsin-like serine protease, and a pH- and salt-resistant protein. The N-terminal amino acid sequence of the enzyme showed similarity with those of trypsin from other organisms. The molecular weight and isoelectric point of the enzyme were estimated to be 38, 000 and 4.65, respectively. The enzyme is probably useful as a thrombolytic agent.

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