The purpose of this paper is to elucidate the final degradative pathways of atrazine (I) by a Nocardia strain through experiments on the degradation of its metabolite, 4-amino-2-chloro-6-isopropylamino-1, 3, 5-triazine (IIa). This compound, in the bacterial medium, was transformed into several products identified as 2-chloro-4, 6-diamino-1, 3, 5-triazine (IIe), 4-amino-2-hydroxy-6-isopropylamino-1, 3, 5-triazine (VIII), 4-amino-1, 2-dihydro-1, 3, 5-triazin-2-one (IV) and dicyanodiamidine (VII). The formation of (IIc) was attributed to a microbial N-dealkylation, while the detection of (IV) and (VII) was attributed to a chemical degradation of the intermediate 4-amino-2-chloro-1, 3, 5-triazine (III). Compound (III) undergoes rapid hydrolysis and does not accumulate in the culture medium. On the basis of the microbial and chemical results, we propose a degradative pathway for atrazine.

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The major protein antigens of the yeast cell wall, TLA a and TLA b, were found to specifically bind with the alkali-soluble fraction (ASF) which was prepared from yeast cells (Saccharomyces cerevisiae) essentially by the method of Manners et al.
Also, TLA a and TLA b were found to be present in the alkali-soluble fraction of yeast cells. This fraction was prepared from yeast cell homogenates after solubilizing by alkali (pH 9.0) followed by precipitating with acid (pH 6.0).
The binding of TLA a and TLA b with ASF was completed rapidly. The optimum pH for the binding was 6.0. Concentrations of sodium chloride higher than 0.06 M and 0.1M inhibited the binding of TLA a and TLA b, respectively. TLA a or TLA b-ASF complexes were dissociated commpletely in 0.2M and 0.6M NaCl solution, respectively.
ASF was reconstituted on the surface of protoplasts after 3 hr of incubation.

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Bacillus sp. No. 106, which was isolated from soil, secreted an enzyme that hydrolyzed cyclo(Gly-Gly). The enzyme was purified to the ultracentrifugally homogeneous state and an activity more than 450-fold that of culture broth. The enzyme was activated by Na⁺, Mg²⁺, Ca²⁺, and Sr²⁺, and strongly inhibited by Ni²⁺, Cu²⁺, p-chloromercuribenzoate, and monoiodoacetic acid. The Km value for cyclo(Gly-Gly) was estimated to be 11.1 mM. The enzyme hydrolyzed only cyclo(Gly-Gly) among various diketopiperazines tested. Aslo, the enzyme was inert toward Gly-Gly, milk casein, and hemoglobin.

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Multiple forms of neutral α-glucosidase (pH optima, 6.0-6.5) were purified from pig duodenal mucosa by a procedure including Triton X-100 treatment, fractionation with ammonium sulfate, fractionation with ethyl alcohol, DEAE-cellulose column chromatography and preparative polyacrylamide disc gel electrophoresis. All of the α-glucosidases, I_a, II_a, I_b and II_b, were found to be homogeneous on polyacrylamide disc gel electrophoresis. The molecular weights, isoelectric points and optimum temperatures of α-glucosidases I_a and II_a were 145, 000-150, 000, pH 3.5-3.7 and 55°C, respectively, and both enzymes were stable up to 55-16 on treatment at pH 6.0 for 15min; whereas those of the other two α-glucosidases, I_b and II_b, were 80, 000, pH 4.0 - 4.1 and 65°C, respectively, and both enzymes were stable up to 70°C on the same treatment. The Km values of enzyme II_a for maltose, maltotriose and amylose were 1.72mM, 0.37mM and 1.67mg/ml, while those of enzyme II_b were 3.33 mM, 2.61 mM and 11.8mg/ml, respectively. All enzyme hydrolyzed α-, 1-4- α-1, 3- and α-1, 2-glucosidic linkages in substrates, but showed no activity on sucrose or isomaltose. Enzymes II_a and II_b hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside, and maltotriose was formed as the main α-glucosyltransfer product from maltose. It was revealed that two types of neutral α-glucosidases having no activity toward sucrose or isomaltose existed in pig duodenal mucosa, and that one type comprised α-glucosidase having both maltose- and amylaceous α-glucan-hydrolyzing activities and the other type heat-stable maltooligosaccharidases which hydrolyzed amylaceous α-glucan weakly.

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The neutral fraction of clove essential oil was studied using silica gel fractionation and Girard T reagent extraction. The four fractions obtained with the first method according to odor assessment contained successively terpenic and sesquiterpenic hydrocarbons, carbonyls and esters, sesquiterpenic alcohols and a lactone.
18 compounds were identified by mass spectrometry and retention time determination or PMR: α-tujene, β-muurolene, β-selinene, cubenene, hexanal, 2-hexanone, 6-methyl-5-heptene-2-one, fenchone, geranial, furaldehyde, 5-methyl furaldehyde, cuminaldehyde, acetophenone, methyl palmitate, methyl stearate, methyl linoleate, benzyl benzoate and γ-decalactone, and 3 were tentatively identified by mass spectrometry: propyl benzoate, benyl salicylate and palustrol.

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The crude fraction extracted at pH 6.0 from sprouting potato tubers (pH 6.0 fraction) hydrolyzed casein and BANA at pH 6.0. This pH 6.0 fraction contained not only caseinase activity but also gelatinase activity, detected by active staining of PAGE-gel with gelatin, as endopeptidases, and both activities increased during sprouting of tubers. This endopeptidase, also active on Azocolase, had an optimum pH at pH 6.0, whereas the crude fraction extracted at pH 6.0 from fresh potato tubers contained little endopeptidase activities in the whole pH range. Inhibition by monoiodoacetate or antipain indicated this endopeptidase to be a cysteine protease.

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Proteinase A₂, a chymotrypsin type enzyme, was purified about 280-fold from an extract of Euphausia superba by DEAE-cellulose column chromatography, gel filtration on Sephadex G-75, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was 29, 000. The optimum pH was 8.0 and the optimum temperature was 45°C.
Proteinase A₂ hydrolyzed casein but not benzoyl-DL-arginine-p-nitroanilide, unlike other serine proteinases from antarctic krill.¹⁾ This enzyme was inhibited by DFP, PMSF, soybean trypsin inhibitor, but not TLCK, leupeptin, or antipain. Proteinase A₂ could hydrolyze benzoyl-tyrosineethylester and was inhibited by chymostatin.
These results suggest that proteinase A₂ from antarctic krill seemed to be a new type of anionic chymotrypsin.

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The milk-clotting enzyme fraction from Irpex lacteus (IR) was obtained by affinity chromatography. To evaluate IR as a calf rennet substitute, Cheddar cheese-making trials were done. There was no difference in cheese yield, protein recovery, or fat recovery between cheese made with calf rennet (CR) and that made with IR. Although IR cheese showed a slightly higher extent of proteolysis in comparison to the control during ripening, IR cheese did not develop a bitter taste even after 6 months of ripening. These facts indicate that IR is a promising rennet substitute for cheese-making.

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Milk-clotting enzymes may be classified into two groups according to their degradation pattern of α_s1-casein in solution at pH 6.0. On the one hand, calf chymosin and Mucor miehei enzyme produced only one degradation product corresponding to α_s1-I under the conditions we employed. On the other hand, Irpex lacteus and Endothia parasitica enzymes produced several degradation products accompanied by a product corresponding to α_s1-I. The Irpex milk-clotting enzyme hydrolyzed α_s1-casein at the positions of His(8)-Gln(9), Phe(23)-Phe(24), Lys(103)-Tyr(104), and Phe(153)-Tyr(154). Irpex enzyme has only one common cleaving site with calf chymosin, that is, the Phe(23)-Phe(24) bond of α_s1-casein.

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Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.
Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.
Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.

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A glucoamylase was purified from the culture broth of Paecilomyces varioti AHU 9417 by precipitation with ethanol, chromatography on DEAE-Sepharose CL-6B, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was estimated to be 6.9×10⁴ by SDS-disc electrophoresis, and the optimum pH was 4.5. The enzyme preferentially hydrolyzes α-1, 4-glucosidic linkages in a series of maltooligosaccharides, and is capable to slowly hydrolyzing nigerose, isomaltose, and panose, but is not active on kojibriose. Soluble starch, amylopectin, glycogen, and β-limit dextrin are rapidly degraded. Activity toward raw starches is very low, but rice and waxy corn raw starches are relatively subject to attack. The subsite affinities (A_i) of each subsite (i) in the active site of the enzyme were evaluated from the Michaelis constants (Km) and the molecular activities (k₀) for a series of maltooligosaccharides according to the subsite theory. The active site was considered to be made up of about four subsites: A₁ ?? 0.46 kcal/mol, A₂ ?? 4.78 kcal/mol, A₃ ?? 1.76 kcal/mol and A₄ ?? 0.67 kcal/mol.

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In order to investigate the effect of the spacer in pepstatin-Sepharose on adsorption and elution of acid protease (AcP) in raw shoyu (unpasteurized soy sauce), a homologous pepstatin-aminoalkyl agarose series, (pepstatin-NH₂(CH₂)_n-Sepharose), that varied as to the length of the hydrocarbon chains was synthesized. When raw shoyu containing many kinds of proteases was subjected to affinity chromatography on these pepstatin-C_n-Sepharoses (n=2, 4, 6, 8, 10 and 12), all of them adsorbed AcP. With increasing length of the spacer up to 6, more and more AcP became adsorbed onto the pepstatin-C_n-Sepharose, whereas with decreasing length of the spacer, more and more AcP was eluted with 0.05M acetate buffer (pH 3) containing 2M urea. The AcP was purified in one step from raw shoyu and did not have any carboxypeptidase activity. Some properties of the major component of the eluted AcPs were as follows: molecular weight, 6.7×10⁴, on gel nitration with TSK-G3000SW, optimum pH for activation of trypsinogen, 3.5, optimum pH for hydrolysis of hemoglobin, 2.75, and the Ki value toward pepstatin, 1.0×10^-8M.

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Enzymatically modified proteins (BMP) with different methionine levels were produced from soy protein isolate using an improved plastein reaction. The products having methionine at approximate levels of 4%, 7%, and 14%, designated as EMP₄, EMP₇, and EMP₁₄, respectively, were investigated to characterize their chemical properties particularly in terms of the state and location of the methionine residues. Leucine aminopeptidase treatment of the EMP products did not find any significant amount of methionine residues at the N -terminals, but carboxypeptidase A treatment liberated methionine efficiently in accordance with the methionine levels in the EMP products. Treatment with LiBH₄ reduced the methionine content of EMP₁₄ by approximately 64%. A significant amount of homoserine was produced when EMP₁₄ was treated with BrCN. All these data indicate that the covalently attached methionine molecules are localized at or near the C-terminals of the EMP molecul

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The effect of solar-withering and turn over treatment during indoor-withering on the development of pouchong tea aroma were investigated by comparing the aroma compositions of nine tea samples. Each sample was manufactured by different degrees of solar withering and different times of turn over treatment.
Among 70 identified constituents of the aroma concentrate, 33 compounds were selected for comparing the aroma compositions.
In general, solar-withering and turn over treatment had positive effects on the development of aroma constituents, except for linalool and geraniol, the effect of the turn over treatment being greater than that of solar-withering. The concentrations of hexenyl esters, oxidized products of linalool, aromatic alcohols, phenylacetaldehyde, sesquiterpenes, cis-jasmone, jasmine lactone, benzyl cyanide and indole were increased markedly by turn over treatment, even without solar-withering.
Seventeen minutes solar-withering. and four turn over treatments seemed to be the most effective to produce the elegant floral aroma of pouchong tea.

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Hydrophobic (R, S)-5-acyloxymethyl-3-alkyl-2-oxazolidinones were successfully hydrolyzed stereospecifically with lipoprotein lipase Amano 3 (LPL) adsorbed on Amberlite XAD-7. The LPL immobilized on the resin was not desorbed on washing with phosphate buffer, toluene or hexane. The activity loss of the column was little during storage for five months at room temperature. Stereospecific hydrolyses were performed pulsewise with the column, and separation of the hydrophobic (S)-ester from the hydrophilic (R)-alcohol was also performed on the same column utilizing the hydrophobic interaction between the (S)-ester and the support resin. The immobilized LPL column was stable on repetition of these operations for over 20 times. The optical purities of the obtained (S)-oxazolidinones, which were favorable intermediates for the synthesis of (S)-β-blockers, were 96-98% e.e.

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The Stereochemical inversion of (R)-5-hydroxymethyl-3-tert-butyl-2-oxazolidinone (1a) or (R)-5-hydroxymethyl-3-isopropyl-2-oxazohdinone (1b) to the corresponding (S)-isomer was accomplished via a key intermediate, (R)-3-N-ethoxycarbonyl-N-tert-butylamino-1, 2-epoxypropane (5a) or (R)-3-N-ethoxycarbonyl-N-isopropylamino-1, 2-epoxypropane (5b), in a high enantiomeric excess. (S)-1a (99% e.e.) or (S)-1b (97% e.e.) was thus obtained from the respective (R)-isomer (1a; 99% e.e., 1b; 95% e.e.).

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Circular dichroism spectra in the ultraviolet region, similar to the spectrum of the collagen helix, were regenerated when various grades of industrial gelatin were allowed to stand at 5°C in a concentration low enough that they did not form gels. Spectrum generation was decreased by an addition of salt or alcohol and by raising the temperature. Maximal regeneration took place at the respective iso-ionic points "Of the acid- and alkali-processed gelatins. As judged from these spectra, 60% of the helical structure of collagen was regenerated from the industrial gelatin that had the highest α-chain content.

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Milk powders (commercial skim milk and modified milk powders) were stored at 30°C and 40°C for one month under various water activities (Aw), 0.23-0.82. After storage under Aw 0.57 and 0.80, they turned a dark brown color, and the contents of methionine and lysine had significantly decreased: methionine had been oxidized to methionine sulfoxide and lysine had changed to an unavailable form. The contents of arginine, tyrosine and tryptophan had also decreased, as had the contents of leucine and histidine in the skim milk powder. The tryptic digestibility of the milk powders had markedly decreased, and both the chymotryptic and peptic digestibilities of the skim milk powder had also decreased. The samples lost the property of being clotted by chymosin treatment.
No significant change was observed with casein under these conditions, nor with milk powders under dry conditions and Aw 0.23.

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Oryzalexins A, B and C were isolated as a group of novel phytoalexins from rice (Oryza saliva) blast leaves infected with Pyricularia oryzae. The basis of the structures of Oryzalexins A, B and C was laid by spectroscopic methods and their inhibitory activities against spore germination and germ tube growth of P. oryzae were assayed.

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Oryzalexins A, B and C, isolated from rice leaves infected with P. oryzae as a group of novel phytoalexins, were confirmed to be (+)-sandaracopimaradiene derivatives by chemical and spectroscopic studies, i.e. A: 3-oxy-7-oxo- (I); B: 3-oxo-7-oxy- (II) and C: 3, 7-dioxo-(+)-sandaracopimaradiene (III).

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A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N₂-fixation, N₂-fixing facultative hydrogen autotrophs listed in "Bergey's Manual of Systematic Bacteriology" were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin dirhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N₂-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter autotrophicus.

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The nickel requirement and the role of nickel were investigated in a recently identified oxygen-resistant hydrogen bacterium, Xanthobacter autotrophicus strain Y38. When 0.3μM NiSO₄ was added to the basal medium which had not been supplemented with nickel, the cell concentration of autotrophically grown strain Y38 increased by about 4-fold and the resumption of cell growth occurred in the stationary phase. These results showed the requirement of nickel for the autotrophic growth of strain Y38. Since a trace of nickel was detected in the basal medium, the role of nickel was investigated using 0.2 mM or 0.4 mM EDTA-containing media. Other trace elements, Ca, Co, Cu, Mn, Mo and Zn, could not replace nickel. Nickel was not required for the heterotrophic growth of strain Y38. Nickel seems to be related a little to urease in strain Y38. Moderate hydrogenase induction was observed in hydrogenase deficient cells of strain Y38 under 95%H₂+5%O₂ when 300 μM NiSO₄ was added to 0.4mM EDTA-containing buffer but it was completely inhibited by chloramphenicol, indicating that nickel was related to the hydrogenase synthesis. A nickel dependent increase in growth rate was demonstrated equally under 40%O₂ and 10%O₂, suggesting that nickel was not directly related to the oxygen-resistance of strain Y38.

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A strain of Micromonospora sp. producing a lytic enzyme toward Serratia marcescens was isolated from soil. The lytic enzyme, called 152-enzyme, was purified from the culture filtrate by salting-out with ammonium sulfate, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-75. The molecular weight of 152-enzyme was 17, 000 and the isoelectric point was pH 7.3. The 152-enzyme showed lytic activity toward S. marcescens, Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Bacillus subtilis, but was completely intert toward Staphylococcus aureus. The enzyme also showed caseinolytic activity. The lytic and caseinolytic activities of 152-enzyme were maximum around pH 11.0 and at 60°C. Both activities were inhibited by DFP and API-2c. Liberation of amino groups from cell walls of P. aeruginosa by incubation with 152-enzyme suggested that the enzyme was a kind of cell wall-lytic peptidase.

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Cryostorage is one suitable method to preserve various desired types of cells. However, all cells do not survive after storage in liquid nitrogen. This suggests the possibility that the properties of the cells which survive after the storage differ from those of the unfrozen original cells.
Therefore, we did the same freeze-thaw procedure of cultured green Lavandula vera cells over again and compared the metabolic and the differentiation potentials of the cells which survived after the repeated freeze-thaw procedures with those of the unfrozen original cells. The results we found were that the frequency of colony formation of the cells which survived after the procedures was high, but that the biosynthetic capability for biotin and the differentiation potentials such as chloroplast formation and plantlet formation of the cells were equal to those of the unfrozen original cells. Cryostorage of cells in liquid nitrogen is discussed in terms of the preservation of various desired types of cells.

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The effects of chemical modifications on the antigenic properties of bovine β-lactoglobulin was studied by three different assay systems with IgG antibodies; immuno-double diffusion, quantitative immunoprecipitin assay, and radioimmunoassay. Carboxymethylation, succinylation, and guanidination seemed not to disturb the antigenicity of β-lactoglobulin in immuno-double diffusion, but the decrease in the affinity of these derivatives for anti-β-lactoglobulin antibody was observed in the quantitative immunoprecipitin assay and radioimmunoassay. Nitrophenylsulfenylation or reduction and Carboxymethylation seemed to delete one of the antigenic sites of β-lactoglobulin judging from the data of immuno-double diffusion, but the results from the quantitative immunoprecipitin assay and radioimmunoassay showed that reduction and Carboxymethylation affected the antigenicity of β-lactoglobulin more strongly than nitrophenylsulfenylation did.

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In order to elucidate the mechanism of the alteration of proteins induced by vaporized aldehydes, unmodified and chemically-modified lysozymes were exposed in the solid state to vaporized hexanal at 50°C and 5.8 or 75% relative humidity (RH). On exposure at 75%RH, the unmodified lysozyme exhibited polymerization, browning, loss of solubility, fluorescence production and impairment of lysine, tryptophan and methionine residues. Methionine residues seemed to be oxidized to methionine sulfoxide residues. The polymerization did not proceed at 5.8RH. All the above alterations were almost completely prevented by the removal of oxygen from the reaction cells. Acetylation of lysozyme retarded these alterations fairly well except that the impairment of tryptophan residues was unaffected.
On the basis of all the results it is suggested that at the first step the concerned reaction essentially requires hexanal derivatives such as peroxyhexanoic acid and/or related radicals induced through the reaction with oxygen. The second step seems to consist at least of two routes which are independent of each other and require water. One route is assumed to be an amino-carbonyl reaction involving lysine residues. The other route seems responsible for the attack on tryptophan and methionine residues through oxidation involving the radicals.

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N, N'-Dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane-bound ATPase, strongly inhibited the growth, as measured by an increase in cell number, of Dunaliella tertiolecta. However, this inhibition was reversed by simultaneous application of adenosine 5'-triphosphate (ATP) or adenosine 2'-monophosphate (2'-AMP). Adenosine and adenosine 5'-diphosphate (ADP) were ineffective in restroration of the DCCD-inhibited growth. Gibberellin A₃) A3 (GA₃) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) also reversed the inhibition of DCCD on D. tertiolecta growth, although these plant growth regulators did not promote an increase in cell number.

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We selected high-yield cell lines, using protoplast culture of Lithospermum erythrorhizon cells. Three cell lines having different shikonin productivities were used as parent cells for the selection, and cell lines with high productivity were obtained efficiently in every case. The best cell line had 6.45g shikonin/g inoculum/23 days of production which was almost 1.5 times higher than that of the original cell line. The productivities of protoplast-derived cell lines were distributed widely and their average productivity was similar to the original one. The subculture of such a protoplast-derived cell line for eight months showed that its shikonin productivity was stabler than the original cell line.

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An efficient approach to the asymmetric syntheses of a-methylglutamic acid and α-methylornithine is described. Two chiral reagents, (2'S)-N-(2'-methoxymethylpyrrolidine)-2-isocyanopropionamide 4 and (2'S)-N-(2'-hydroxymethylpyrrolidine)-2-isocyanopropionamide 5, were employed for the asymmetric induction. α-Methylglutamic acid 7 was synthesized by the asymmetric Michael-addition of methyl acrylate to 4 and 5 as the key step. The optical yield of 7 was 10-45% (R-form). α-Methylornithine 12 was also synthesized by the reaction of 4 with acrylonitrile as the key step. The optical yield of 12 was 31.7% (R-form).

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A convenient synthetic method for (±)-malyngolide (1), an antibiotic from the marine blue-green alga Lyngbya majuscula GOMONT, is described. The compound (±)-1 was synthesized from 2-methylglutaric anhydride (starting material) in four steps, involving the TBHP-based osmiumcatalyzed procedure for vicinal dihydroxylation of the methylene unit in unsaturated carboxylic acid as the key step. The optical resolution of (±)-1 was accomplished by HPLC, in which diastereomeric (+)-2-(4-chlorophenyl)isovaleric acid [(+)-CPIA] esters of (±)-1 derived from (+)-CPIA-C1 could be easily separated, and subsequent hydrolysis of each ester gave the enantiomerically pure (-)-1 and (+)-1, respectively. A synthetic method for α-methylene malyngolide, which is to be expected for biological activity, is also described.

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A further investigation of the isoflavonoid constituents occurring in roots of the white lupin (Lupinus albus L. cv. Kievskij Mutant) has yielded five new Coumaronochromones named lupinalbin A (1a), B (2a), C (3), D (4) and E (5). These isoflavonoids were identified by physicochemical methods involving the use of biogenetically related 2'-hydroxyisoflavones as reference compounds. The presence of the rare dihydrofurano-isoflavone, erythrinin C (16), in white lupin roots has also been established.

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In order to investigate the headspace volatiles which indicate the againg effect on tobacco, the headspace volatiles of Japanese flue-cured tobacco were collected by active carbon and analyzed by gas chromatography. The data obtained were subjected to principal component analysis (PCA) and stepwise discriminant analysis (SDA). The results of PCA showed that the volatiles indicated the effect of aging on smoking quality. The SDA selected six peaks of the volatiles which classified the samples into four groups according to the aging period. The results described above can show that the relative proportions of the volatiles, such as furfuryl alcohol, benzyl alcohol and solanone, may be used to judge approximately the agint effects on tobacco.

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Four new bitter terpenoids, lucidenic acids A (1), B (2), C (3) and ganoderic acid C (5), were isolated from the fruiting bodies of Ganoderma lucidum, together with the known bitter ganoderic acid B (4). On the basis of spectroscopic data and chemical conversion, their structures were determined to be 7β-hydroxy-4, 4, 14α-trimethyl-3, 11, 15-trioxo-5α-chol-8-en-24-oic acid, 7β, 12β-dihydroxy-4, 4, 14α-trimethyl-3, 11, 15-trioxo-5α-chol-8-en-24-oic acid, 3β, 7β, 12β-trihydroxy-4, 4, 14α-trimethyl-11, 15-dioxo-5α-chol-8-en-24-oic acid and 7β-hydroxy-3, 11, 15, 23-tetraoxo-5α-lanost8-en-26-oic acid, respectively.

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For Hyphomicrobium 53-49 capable of growing under various conditions, aerobic methanol, anaerobic methanol (with denitrification), autotrophic (H₂-O₂-CO₂), aerobic ethanol and aerobic acetate, investigation and comparison of the specific activities of the following enzymes were performed: alcohol dehydrogenase (NAD-ethanol linked and NAD-methanol linked), primary alcohol dehydrogenase, formaldehyde dehydrogenase (NAD-GSH linked and DCPIP linked), formate dehydrogenase, serine hydroxymethyl transferase, hydroxypyruvate reductase, isocitrate lyase (icl), malate lyase, malate dehydrogenase, ribulosebisphosphate (RuBP) carboxylase, phosphoenolpyruvate (PEP) carboxykinase (ADP linked), PEP carboxylase (phosphorylating), pyruvate carboxylase (NADH linked and NADPH linked) and α-ketoglutarate carboxylase (NADH linked and NADPH linked). On the basis of the data obtained, it was concluded that during growth on methanol, aerobically and anaerobically, the icl⁺ serine pathway operated, while during autotrophic growth on H₂-O₂-CO₂, CO₂ was incorporated through the RuBP pathway and others, and during growth on ethanol or acetate, neither the serine pathway nor the RuBP pathway operated. The organism changed its metabolism through the regulation of the metabolic enzymes according to the growth conditions.

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The mechanism of moisture transfer into shredded tobacco was investigated in connection with moistening process control in cigarette manufacturing. For shredded tobaccos of less than about 1.0mm shred width, the moisture was sorbed mainly through the cut faces rather than through the leaf epidermis, indicating that the moisture diffusion within the leaf was the rate limiting step of moisture transfer. The moisture diffusion coefficient values within the cured tobacco leaf ranged from 8.80×10^-7 to 64.2×10^-7cm²•s^-1 depending on the type of tobacco, the shred width of tobacco and the temperature at sorption. But these values were not affected by the relative humidity at sorption. The entire moisture transfer into the shredded tobacco leaves was predictable with the diffusion theory.

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Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.

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In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β-oxidation-acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase-were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.
The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.

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In order to investigate the production of a strong bitter taste of the tetrapeptide, Arg-Pro-Phe-Phe (1), we synthesized 16 kinds of analogs and tasted them. From the results, it was clarified that all the constituent amino acid residues in Arg-Pro-Phe-Phe (1) were necessary for its strong bitter taste. For a further increase in bitterness potency, it was found that the bitterness production units necessary should be concentrated together. In addition, Arg-Pro-Gly-Gly (6) and Gly-Gly-Arg-Pro (7) were found to have no bitterness. This will be very useful not only for studies on debittering of food but also for basic studies on the taste production mechanism.

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Two streptothricin-like antibiotics, Nos. 6241-A and B, were obtained as inhibitors of de novo starch synthesis in excised leaf segments of barnyard millet (Panicum crus-galli). No. 6241-B was identified as SF-701, and No. 6241-A was a new antibiotic, in which N, N-dimethylglycine was substituted for the N-methylglycine of No. 6241-B (SF-701). Both antibiotics also inhibited plant growth. The inhibitory activity of No. 6241-B (SF-701) was approximately ten times that of No. 6241-A. By foliar treatment, No. 6241-B (SF-701) showed remarkable herbicidal activity against barnyard millet at a concentration of more than 500 ppm with little phytotoxicity for the rice plant (Oryza saliva L. cv. Nihonbare).

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OmpC and OmpF, outer membrane porin proteins, are important in the maintenance of the cell surface structure of Escherichia coli cells [T. Nogami and S. Mizushima, J. Bacterial., 156, 402 (1983)]. Mutants lacking both proteins are unstable and frequently revert or mutate to strains which either have regained one or both of the proteins or constitutively produce PhoE, another porin protein. In the present work, the structural importance of PhoE was studied in relation to OmpC. and OmpF. The strain devoid of both OmpC and OmpF was highly susceptible to Tris-HCl buffer at a concentration of 120mM in terms of viability and cell structure. This strain was also susceptible to osmotic shock. In contrast, the strain possessing PhoE in place of OmpC/OmpF was as stable as the strain possessing OmpC/OmpF against these treatments. PhoE, like OmpC and OmpF, was assembled into a hexagonal lattice with lipopolysaccharide that covered the peptidoglycan sacculus. These results suggest that PhoE can take the place of OmpC/OmpF in the maintenance of the cell surface structure. The importance of porins in general in the maintenance of the cell structure is discussed.

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