Two new guaianolides combined with a sulfur-containing acetylenic compound were isolated from Arctium lappa (Compositae). Their structures were elucidated on the basis of spectral data.

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Endo-β-N-acetylglucosaminidase, purified to homogeneity from the culture nitrate of a Flavobacterium sp., liberated the carbohydrate chains from yeast invertase. About 90% of the carbohydrate associated with this glycoprotein was removed by the endo-β-N-acetylglucosaminidase. The native and carbohydrate-depleted enzymes were compared and found to exhibit similar catalytic activities, thermal stabilities and pH-activity profiles. However, the carbohydrate-depleted invertase was more susceptible to proteases with relatively broad specificities such as subtilisin and pronase, as found on examination of the enzyme activity and electrophoresis. On the other hand, trypsin did not have such an effect on the enzyme activities of the native and carbohydrate-depleted enzymes.
The endo-β-N-acetylglucosaminidase also released carbohydrate chains from the purified β-N-acetylhexosaminidase of Penicillium oxalicum. Although the native and carbohydrate-depleted β-N-acetylhexosaminidases did not differ significantly in their stabilities or pH-activity profiles, the carbohydrate-depleted form was more susceptible to proteolysis by subtilisin, pronase and trypsin. From these results, it would appear that the carbohydrate of a glycosylated enzyme plays a role in protecting the enzyme from proteolysis.

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A gene cluster from the chromosomal DNA of Pseudomonas putida encoding the oxidation of benzene to catechol was cloned into a broad-host-range cosmid vector, pMMB33. Escherichia coli cells containing these cloned genes were able to accumulate catechol from benzene and showed green pigmentation, presumably, due to the accumulation of indigo produced by benzene oxidizing enzymes. A mutant strain, 81CB46, of P. putida, which is deficient in the oxidation of benzene to catechol, gained the ability to grow on benzene due to the cloned genes. The functional region of the cloned fragment was less than 5 kb in length.

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A new enzyme that catalyzes the dehydration of meso-tartrate was found in the cell-free extract of Pseudomonas putida isolated from soil. The bacterial cells also produce D-tartrate dehydratase which acts on D-tartrate specifically. The meso-tartrate dehydratase is inducibly produced on the addition of meso-tartrate to the medium, and is very unstable. The enzyme is specific for meso-tartrate, other isomers not acting as substrates. The enzyme requires no cofactor, although it is inhibited by oxygen and thiols. The properties of the enzyme differ from those of D- and L-tartrate dehydratases from pseudomonads reported previously.

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The peptic hydrolysis of bovine β-lactoglobulin (β-Lg) was performed to establish the basis for producing a low-phenylalanine peptide rather than a free amino acid mixture for use in the dietetics of phenylketonuria. A 1% β-Lg solution (pH 1.5) was incubated with 0.01% pepsin at 37°C for 24 hr. The peptides produced were fractionated by high-performance liquid chromatograhy to analyze their constituent amino acids. Most of the major peptides were identified in the light of the primary structure of α-Lg to assign 31 cutting points in their protein molecule. These included cutting points at thecarboxyl side of Phe-82, Phe-105 and Phe-136. This result suggests that further hydrolysis of the peptic hydrolysate of β-Lg with an exopeptidase, particularly with a carboxypeptidase, would be effective in liberating phenylalanine to produce a low-phenylalanine peptide mixture.

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A proteinase found in rice seeds during germination was purified by (NH₄)₂SO₄ fractionadon, CM-Sephadex chromatography, Sephadex G-100 gel filtration, Phenyl-Sepharose CL-4B chroraatography, and chromatofocusing. The purified enzyme had a molecular weight of about 23, 000 and an isoelectric point at pH 5.15. Its optimum pH for hydrolysis lay at 5.5 and at 5.75 when measured with α-N-benzoyl-DL-arginine-2-naphthylamide and casein, respectively. The proteinase was activated by thiol compounds and it was inhibited by common thiol-blocking reagents, particularly by leupeptin, antipain, and E-64. It was also inhibited, in a non-competitive manner, by a cysteine proteinase inhibitor purified from rice seeds. These results suggests that this enzyme belongs to the cysteine proteinase class (EC 3.4.22).

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From the aerial part of Bupleurum kunmingense Y. Li et S. L. Pan, three new saikosaponin derivatives named BK1, BK2 and BK3 were isolated and their structures determined as 3-O-β-glucopyranosyl-(1→6)-(α-rhamnopyranosyl-(1→4))-β-glucopyranoside of 16-epi-saikogenin C, 3-O-β-glucopyranosyl-(1→6)-(α-rhamnopyranosyl-(1→4))-β-glucopyranoside of 30-hydroxy-16-epi-saikogenin C, and 28-O-β-glucopyranosyl-3-O-β-glucopyranosyl-(1→6)-(α-rhamnopyranosyl(1→4))-β-glucopyranoside of 16-epi-saikogenin C, respectively.

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In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.

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The microbial production, by the genus Rhizopus, of a gaseous saturated-hydrocarbon mixture was studied under aerobic conditions. Rhizopus strains, comprising 13 strains of 9 species, were tested as to their ability to produce a gaseous hydrocarbon mixture. Except for one strain, all the strains tested produced more than two kinds of gaseous hydrocarbons simultaneously when grown in nutrient broth containing glucose. Rhizopus japonicus IFO 4758 was selected as being typical of these producers of mixed gaseous hydrocarbons. When this organism was cultivated in a synthetic medium supplemented with L-cysteine under aerobic conditions, the maximum production of the total gaseous hydrocarbon mixture reached ca. 10 nl/ml culture broth/hr. The gaseous hydrocarbon mixture produced was composed mainly of paraffin hydrocarbons, i.e., ca. 74% pentane, ca. 16% propane and a trace amount of methane. The ratios of saturated to unsaturated, and even to odd number hydrocarbons produced by this fungus were 95:5 and 90:10, respectively. The biosynthetic pathways for the production of these gases are discussed in comparison with the biosynthetic pathways for ethylene and isobutene in microorganisms.

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β-CN(f 193-209), a hydrophobic peptide of 17 residues obtained from the chymosin hydrolyzate of β-casein, had little emulsifying activity (EA) at a neutral pH. When mixed with a hydrophilic glycomacropeptide (GMP) derived from κ-casein however, the EA of β-CN(f193-209) increased greatly. The mixing ratio of the peptides affected the EA as well as the adsorption of the peptides to oil droplets. Scanning electron microscopy indicated that the peptide film surrounding the emulsified oil droplets was thick and rough compared to the protein film. An amphipathic structure formed by some interaction between the hydrophilic GMP and the hydrophobic β-CN(f193-209) might contribute to the formation of the thick peptide film and stabilize the emulsified oil.

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A novel yeast-lysing bacterium (YLM-1) grew on nutrient agar containing yeast cell-free extract, but not on nutrient agar. We searched for the growth factor for YLM-1, which turned out to be hemin or hemoproteins, catalase or horseradish peroxidase. Also, the effects of growth accelerators and inhibitors on H₂O₂ metabolism were discussed.

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The antifungal activity of 441-acyl derivatives of 3-(3, 5-dichlorophenyl)-2, 4-imidazolidinedione against Botrytis cinerea, and of 10 l-sulfonyl compounds against Alternaria kikuchiana were assayed by the agar medium dilution method. The structure-activity relationships for the substituents of the acyl and sulfonyl moieties were analyzed with such physicochemical parameters as hydrophobic π, inductive electronic σ₁, and steric E^'c_s and B₁ values by multiple regression. The activity of the acyl derivatives against B. cinerea was related parabolically to the hydrophobicity of the substituents. The stronger the electron-donating power, the larger the overall steric bulkiness, and the smaller the minimum width in the direction perpendicular to the bond axis of the substituents, the greater was the activity. The activity of the sulfonyl derivatives against A. kikuciana was related only to the hydrophobicity of the substituents.

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Malate synthase (EC 4.1.3.2), termed MS_H (mol. wt. 630, 000), was purified from the pollen of Pinus densiflora Sieb. et Zucc. to apparent homogeneity as judged by SDS-PAGE. Part of MS_H was converted to the low molecular weight form of MS, termed MS_L (mol. wt. 62, 000), by incubation with 5 mM ATP. Both forms of MS were maximally active at pH 7.6 in the presence of 10 mM MgCl₂ and 0.01mM EDTA. MS_L was completely inactivated by heating at 50°C for 3min, but MS_H activity was retained about 65%. MS_H preincubated with 5mM ATP showed unstability similar to that of MS_L. MS_H activity was more strongly inhibited than that of MS_L by phosphoenolpyruvate or ATP. Km values of MS_H for acetyl-CoA and glyoxylate were about one half of those of MS_L. The V_max of MS_H was about 5 times as high as that of MS_L. ATP was a noncompetitive inhibitor with respect to acetyl-CoA and Ki values of ATP for MS_H and MS_L were 1.0 × 10^-3 M and 2.0 × 10^-2 M, respectively. From these results we suggested that MS_H and MS_L are the active and low active forms of MS, respectively.

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The sizes and shapes of lutein aggregates in acetone aqueous solution were examined by spectroscopic analyses and electron microscopic observation, since lutein dispersed into acetone aqueous solution and provided a much simpler system than others. This system is suitable for basic research on lutein aggregates. The lutein aggregate gave maximum molar ellipticity in acetone concentrations from 35 to 45% aqueous solution. When the acetone concentration increased, CD spectra were reversed in 45% concentration from a negative exciton chirality to the positive. Since the aggregates were most slender in these acetone concentrations, it was found that the length of the lutein aggregate was closely related to the molar ellipticity. The apparent widths of the aggregates became large with increases of the acetone concentration after the reversion of CD spectra, where the molar ellipticities were diminished. These results are similar to those for the lutein aggregates in dilute surfactant solution, and this system is clearer and simpler for electron microscopic observation than those in surfactant solutions.

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An electron microscopic study was done on lutein aggregates dispersed in liposomes of egg yolk PC and DGDG of spinach leaves. A left handed helical structure of the lutein aggregate, which was expected from the CD pattern of negative exciton chirality, was observed in PC liposomes of high pH in the presence of Ca²⁺. The lutein aggregate in the PC liposome gave fine long images which were 20 to 40nm wide and more than 2000 nm long. The aggregates became longer with an increase of the PC concentration. Similar helical structures of lutein aggregates were observed in the DGDG liposomes.

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During research on the production of human α-interferon (α-IFN) in silkworms using a baculovirus, Bombyx mori nuclear polyhedrosis virus (BmNPV), as a vector, the expression of approximately 5.0×10⁷ U/ml of α-IFN in the haemolymph was attained. In order to obtain a much higher level of expression, we paid special attention to the role of the sequence between the promoter and the translation start site of the polyhedrin gene, and found that deletion of the sequence led to depression of the expression. We therefore tried to improve the cloning vector. By means of the in vitro mutagenesis technique, a cloning vector retaining this sequence and having various restriction enzyme sites between the promoter and the terminator of the polyhedrin gene was constructed. Using this vector, a transfer vector was constructed in which the polyhedrin structural gene was replaced by human α-IFN, and recombinant viruses were prepared. When silkworms were infected with the recombinant viruses, about 1.9×10⁸ U/ml of IFN was produced in the haemolymph of fifth instar larvae. The improvement of the vector raised the productivity of IFN approximately four-fold.

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The fragment-peptides of angiotensin I-converting enzyme inhibitors (CEI₁₂, CEI₅ and CEI_β7) derived from an enzymatic hydrolysate of casein were synthesized. Val-Ala-Pro, the C-terminal tripeptide of CEI₅ (Phe-Phe-Val-Ala-Pro), exhibited more potent inhibitory activity (I₅₀=2.0μM) than CEI₅ (I₅₀=6.0μM). However, D-Val-Ala-Pro showed lower inhibitory activity (I₅₀=550μM). Val-Ala-Pro may be important for the inhibitory activity of CEI₅. The C-terminal heptapeptide of CEI₁₂ (Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys) and the short fragments of CEI_β7 (Ala-Val-Pro-Tyr-Pro-Gln-Arg) showed lower inhibitory activities than the full-length peptides.
Also, the antihypertensive activity of CEI₁₂ and CEI₅ was investigated. CEI₁₂, intravenously administered to anesthetized rats at 14.2mg/kg, antagonized the rats' pressor response to angiotensin I.

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A DNA region exhibiting ARS activity in Candida maltosa was subcloned from an isolated 3.8kb DNA fragment and designated as the TRA region previously [M. Takagi et al., J. Bacterial., 167, 551 (1986)], which replicates autonomously not only in C. maltosa but also in Saccharomyces cerevisiae. It was found that a DNA fragment of about 200bp (designated as fragment 2) exhibited ARS activity in both yeasts, and nucleotide sequence analysis of this fragment revealed that it contained five 11 bp-sequences which are homologous to the consensus sequence of ARS sites of S. cerevisiae [J. R. Broach et al., Cold Spring Harbor Symp. Quant. Biol., 47, 1167 (1982)] (9 among 11 bp being identical in each).
Plasmids constructed using fragment 2 (pTRA2 and pTRA12) will be useful as cloning vectors, although they showed lower transforming frequencies and lower stabilities in C. maltosa than plasmids containing the TRA region (pTRA1 and pTRA11).

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The polymerization and depolymerization of inulin during aging of tubers of Jerusalem artichoke (Helianthus tuberosus L.) was analysed by GPC and HPLC methods. Depolymerisation of inulin as a function of external stress, particularly from drought and frost, was observed. The potential consequences of such changes to the physiology of the tubers and the potential use are discussed. Spring harvested tubers containing low-molecular weight inulin are well suited for fermentations, or for the isolation of inulooligosaccharides. Tubers not exposed to frost and containing high molecular weight inulin are better sources for the production of high-fructose syrup.

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The effects of dietary glutathione (GSH) on plasma and liver lipid concentrations were investigated with rats fed on a high cholesterol diet. When graded levels of GSH, 0.75 to 5.0%, were added to the 25% casein basal diet, the plasma total cholesterol level was significantly decreased and the HDL-cholesterol level was inversely increased in all addition levels without influence on the growth of animals except for the 5% addition level; the dietary addition of 5% GSH markedly depressed the growth and food consumption of rats and caused a slight diarrhea. Plasma triglyceride and phospholipid levels were decreased by the dietary addition of GSH. The contents of cholesterol and triglyceride in the liver were decreased as the dietary addition level of GSH was increased. The dietary addition of a mixture of glutamic acid, cysteine and glycine, or cysteine alone corresponding to 2.5% GSH resulted in a cholesterol-lowering effect which could not be distinguished from the effect of GSH in rats fed on the 25% casein diet. When 1.5% GSH was added to a low (10%) casein diet, the plasma cholesterol-lowering effect of GSH was also observed and the effect was comparable to that of cysteine. These results indicate that dietary-added GSH has a plasma and liver cholesterol-lowering efficacy and that this effect is largely attributable to the cysteine residue of GSH rather than to the tripeptide itself or the other amino acid residues.

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The addition of phenol (about 1 %) to 6 M HCl largely prevented destruction of tryptophan during hydrolysis of peptides at 110°C for 22hr. Tryptophan recovery depended on the volume of 6 M HCl containing phenol and the concentration of phenol. The maximum tryptophan recovery was 85% for a standard amino acid mixture. The recovery was slightly lower for proteins. This hydrolytic procedure was advantageous for micro amino acid analysis using a conventional highperformance liquid chromatography with a precolumn labeling technique. The method was used in the amino acid sequence analysis of a minor component of sea anemone toxins isolated from Anthopleura fuscoviridis. The toxin consisted of 48 amino acid residues with three tryptophan residues.

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An enzyme useful for preparing semisynthetic polymyxins was found to be produced by a Pseudomonas sp. strain, M-6-3 (closely related to Ps. acidovorans). This enzyme, tentatively named polymyxin acylase, is the first enzyme known which can remove the acyl moiety from an acyl peptide without affecting the peptide moiety. It can be produced in a cell-bound form in excellent yield in a medium containing citric acid as the sole source of carbon. The activity of the acetonedried cell powder of the bacterium was optimum at pH 7.5 and remained stable up to about 50°C for 30min. The apparent V_max and Km values for colistin (polymyxin E) were 8.7 nmol/min/mg and 2.8mM, respectively. The suspension of the cell-bound enzyme in colistin solution gave a good yield of deacyl colistin and fatty acid(s), and no chemical change occurred in the peptide moiety during the enzyme reaction. This enzyme deacylated not only polymyxins B, M, and P, but also several other fatty acyl peptides. It should be valuable as a tool for the deacylation of various acyl peptides.

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From immature seed of Phaseolus vulgaris, a new brassinosteroid, 6-deoxodihydrohomodolichosterone, as well as dolichosterone and brassinolide have been identified in addition to 6-deoxodihydrodolichosterone, 6-deoxodihydrocastasterone, castasterone and dolicholide which had already been reported therein. Extensive GC/MS analysis revealed that the number of brassinosteroids fully or partially characterized from the seed totals thirty, including unknown brassinosteroids.

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Carboxylic acids with a cleaved type of cyclopropane ring between the C-2 and C-3 bond in tetramethylcyclopropanecarboxylic acid, the acid part of fenpropathrin, were prepared. Several of their esters with pyrethroidal alcohols exhibited good insecticidal activity, but their activity was a little weaker than that of fenpropathrin.

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Zygosaccharomyces rouxii contains at least two copies of the glyceraldehyde-3-phosphate dehydrogenase gene (GAP-DH). We cloned one of them, which is situated on a 9.5kb HindIII fragment, after detecting a sequence which is hybridizable with the GAP-DH gene of Saccharomyces cerevisiae. The amino acid sequence of the GAP-DH gene of Z. roxii deduced from its nucleotide sequence was compared with that of the GAP-DH genes of S. cerevisiae. The Z. rouxii enzyme is longer than the S. cerevisiae enzyme by one amino acid. 65, 66 or 71 amino acid changes were detected, depending on which GAP-DH gene of S. cerevisiae was compared with that of the Z. rouxii enzyme. The codon usage in the coding region of the GAP-DH gene of Z. rouxii is biased, as found in the case of the S. cerevisiae enzyme.

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An efficient method for the liberation and regeneration of L. edodes protoplasts was developed using an enzyme mixture of Cellulase Onozuka RS and chitinase, pH 4.6. Homogeneous young mycelia as the protoplast material were quickly cultured in a liquid medium with 'wood components' after inoculation of cut fragments of hyphae. The preparation of more than 6 × 10⁷ protoplasts/ml/4hr and regeneration of more than 15% were attained. The dikaryons formed through the mating of monokaryotic strains of protoplast origin could develop complete fruit bodies on small scale sawdust cultivation (30g medium) in 50-60 days. After protoplasting or fusion treatment with polyethyleneglycol, the mating phenotypes were sufficiently well preserved.

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We studied the effects of peptidylarginine deiminase, a Ca²⁺-dependent protein-modulating enzyme that catalyzes the deimination of arginyl residues in protein on two major trypsin inhibitors in mouse plasma. One of these inhibitors was α-1-antitrypsin and the other was a recently characterized trypsin inhibitor termed contrapsin (H. Takahara and H. Sinohara, J. Biol. Chem., 257, 2438 (1982)). The enzyme abolished the trypsin-inhibiting activity of contrapsin in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 3.4 × 10³M^-1•s^-1), but no detectable changes in the activity was noted for α-1-antitrypsin. Millimolar Ca²⁺ and dithiothreitol were absolutely required for the inactivation of contrapsin by peptidylarginine deiminase. Although no significant alterations of the charge and the size in modified contrapsin were observed, the modified inhibitor indicated that 1 arginyl residue was converted to a citrullyl residue. Modified contrapsin whose anti-tryptic activity was lost inhibited chymotrypsin much more strongly than the native inhibitor. These data suggest that a vital amino acid residue for the anti-tryptic activity of mouse contrapsin is an arginyl residue and the conversion of this arginyl residue to a citrullyl residue causes the functional changes of the inhibitor.

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We have isolated a tetracycline-resistant (Tc^r) Bacillus species (named HE-1) which carries multiple plasmids. HE-1 was identified as Bacillus cereus and found to bear four plasmids. Tetracycline resistance could be attributed to one of four plasmids (designated as pTITβ2 (4.7 kb)) indistinguishable from pBC16, a Tc^r plasmid formerly found in B. cereus [K. Bernhard, H. Schrempf, and W. Goebel, J. Bacterial, 133, 897 (1978)]. All the other three plasmids (named pTITα (4.0kb), pTITβl (4.7kb) and pTITγ (12.4kb)) were cryptic and did not correlate with bacterial phenotypic traits such as antibiotic resistance or antibiotic and bacteriocin production. B. cereus HE-1 also showed resistance to penicillin, but this seemed very likely to be chromosomally determined in B. cereus. Of interest was the fact that pTITα, pTITβl, and pTITγ had a noticeable DNA homology among them in blot hybridization. pTITβ2 alone did not shared sequence homology with the other three plasmids.

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