The absorption and excretion of melanoidins, a mixture of the low- and high-molecular weight components (LMM and HMM), and the LMM component prepared from a L-lysine-D-glucose system, at pH 7.4 and 9.0, respectively, and the effects of these melanoidins on cholesterol metabolism were examined in rats. Weanling male rats of the Wistar strain weighing about 50 g were fed diets containing 10% casein (IOC) with 3% of a melanoidin or 25% casein (25C) with 4% of the melanoidin for 12 weeks, and the 25C diet including 3% of the melanoidin or LMM for 8 weeks. The growth and organ weights of the melanoidin-supplied groups were not different from those of the control ones. In rats given the melanoidin diets with both protein levels, the kidneys became dark brownish due to the accumulation of the melanoidin, though the accumulated amount was extremely small (nearly 0.5 mg/g wet kidney), and gel filtration chromatography of a water extract of the kidneys on Sephadex G-75 showed that the deposited melanoidin was the LMM component. Most of the ingested melanoidin, however, was excreted in the feces, and on comparison of the gel chromatographic patterns, the melanoidin groups were found to have more fecal LMM than the control ones. When rats were provided with LMM, HMM increased in their feces. The addition of melanoidin suppressed the level of plasma total cholesterol and elevated the fecal excretion of total lipids and total cholesterol. The urinary contents of protein and total creatinine did not differ from those in the control groups.

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A compound containing fatty acids was detected in the residual fraction of Selenomonas ruminantium cells from which lipopolysaccharide and phospholipid had been extracted. This compound, designated as material M, showed a different mobility from those of lipopolysaccharide and phospholipid on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since 2-³Hglycerol was not significantly incorporated into this material and protease treatment did not affect its mobility on electrophoresis, it is not likely to be a lipoprotein-like compound. Material M was mainly localized in the inner membrane of this bacterium. Material M was purified from the phenol layer which formed on the treatment of phospholipid-deprived cells with phenol-water. The purified material M was estimated to be approximately 95% pure. The major components of the purified M, glucosamine and phosphate were detected in a molar ratio of 2:1. The fatty acid content of material M was only one-tenth that of glucosamine on a molar basis. The fatty acid composition of material M was different from those of lipopolysaccharide and phospholipid. β-Hydroxy fatty acids, which are consistently present in lipopolysaccharide, were not detected in material M.

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The effects of six trace metals on the growth of five species of phytoplankton were examined and their uptake of these metals was investigated. Chattonella antiqua was sensitive to high cupric ion activities with a prolonged lag phase when the activity was 10^-12.8M. The growth of Gymnodinium nelsoni was slowed by low manganese activities and stopped at the activity of 10^-11 M. Thalassiosira sp., C. antiqua, and Heterosigma akashiwo were affected by low zinc ion activities and H. akashiwo was affected by low cupric ion activities. All species but Dunaliella sp. were affected by low iron concentrations. The cellular copper level of Dunaliella sp., the cellular manganese level of Thalassiosira sp., and the cellular zinc level of H. akashiwo were saturated. Thlassiosira sp. could accumulate about 32 times as much manganese as its minimum cell requirement.

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Transglutaminase is a Ca²⁺ -dependent enzyme that covalently polymerizes proteins through the formation of ε-(γ-glutamyl)lysyl cross-links. We have reported that highly concentrated protein solutions were firmly gelatinized by transglutaminase. The technique of gelation of an α_s1-casein solution with transglutaminase was applied to the preparation of an α_s1-casein film. The α_s1-casein film obtained showed a high tensile strength (105 g/cm²) and strain (72%). It was insoluble in water, 10% 2-mercaptoethanol, 6.6 M urea, 10% SDS and 6M guanidine hydrochloride. Even if it was diluted with water and then heated at 100°C for 10min, it remained insoluble. However, it was gradually hydrolyzed by chymotrypsin. These results suggest the usefulness of the α_s1 -casein film as a supporting material of immobilized enzymes, a medical polymer and an edible film.

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An α_s1-casein film prepared using transglutaminase was applied as a support for immobilized enzymes. That is, the enzyme, to be immobilized was added to a mixture of 5% α_s1-casein and transglutaminase. Before gelation by means of the transglutaminase-reaction, the reaction mixture was quickly spread on a horizontal plate. The immobilized enzyme film was removed from the plate after air-drying. Attempts were made to immobilize several enzymes, such as β-glucosidase, α-amannosidase, β-galactosidase and glucose oxidase. None of the immobilized enzymes lost activity on repeated usage. The enzymes tested were evidently immobilized through entrapment in the lattice of the protein film. Some enzymic characteristics of the immobilized enzymes showed that this new technique was as good as other known immobilization methods.

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The chemical constituents of a Japanese sedge "birodo-suge" [Carex fedia Nees var. miyabei (Franchet) T. Koyama] were investigated, and four 3, 5, 4'-trihydroxystilbene (resveratrol 1) oligomers, one dimer, one trimer and two tetramers, were isolated. Their structures were determined by spectroscopic evidence and biogenetic consideration. The dimer was identified as ε-iniferin (2) that had already been identified as a phytoalexin of grape vine leaves. The trimer and the tetramers were structurally new compounds, and were named miyabenols C (for the trimer 5), and A and B (for the tetramers 3 and 4 respectively). The antimicrobial test of 3 (the predominant constituent) revealed that 3 was antibiotic only against Gram positive bacteria.

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The title compound 2 was synthesized. Asymmetric epoxidation of 9 followed by reductive cleavage of the epoxide 10 and acidification gave a symmetrical spiroketal (3), to which pyrrole and benzoxazole moieties were introduced in an efficient manner by the previously developed method. Since the synthetic material was less stable than natural calcimycin under basic conditions, cleavage of the methyl ester was achieved with LiI-pyridine.

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We examined the production of 2-methylisocitric acid, an intermediate of the constitutive methylcitric acid cycle involved in propionyl-CoA oxidation during the catabolism of different amino acids by a mutant lacking 2-methylisocitrate lyase, a key enzyme of the cycle. The acid was produced equimolarly from isoleucine within a range of amounts of the amino acid added. The amount of the acid produced also increased depending upon the amounts of valine, threonine, methionine, homoserine, and α-aminobutyric acid. However, only a little acid was produced from the 13 other amino acids tested. These results indicated that propionyl-CoA was involved in the catabolism of the first six amino acids named, but not in the other 13. Intracellular amino acids were therefore part of metabolic turnover and the constitutive cycle functioned in the catabolism of propionyl-CoA derived from the turnover.

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The ability of rats to regulate branched-chain amino acid intakes was investigated by a self-selection feeding method. The relationship among the consumption of a branched-chain amino acid (BCAA), valine (Val), leucine (Leu), or isoleucine (Ile) and the amino acid concentrations in plasma and brain were also studied. When weanling rats were offered a choice of two diets containing different level of Val, Leu, or He, they consumed Val, Leu, or He ranging from 0.53 to 2.07%, from 0.74 to 3.58%, and from 0.50 to 2.96% of the feed ingested, respectively. The amino acid concentrations in plasma and brain of the self-selecting rats were within a narrower range than those in the fixed feeding rats.
From these results it became clear that when rats were allowed to select their feed, they could regulate Val, Leu, or He intake to meet their requirement for the L-amino acid, and that these amino acid concentrations in plasma and brain were maintained within a narrow range.

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The intracellular factors which may regulate cAMP binding to the inner surface of membranes in human erythrocytes were investigated. [³H]cAMP binding studies on isolated membranes at 30°C in the presence of plausible cAMP binding regulators such as proteins, nucleosides, and non-cyclic nucleotides suggested that ATP could be the most potent regulator of cAMP binding to human erythrocyte membranes. ATP inhibition of [³H]cAMP binding to human erythrocyte membranes was competitive and temperature-dependent. The apparent dissociation constant for the cAMP-membrane complex was 10 nM at 30°C in the absence of ATP. The presence of 1 mM ATP increased the constant to 63 nM.

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An isopropenyl (= 3, 3-dimethylallyl) 3-methoxyflavone (1) and its hydrate (5) were isolated from the roots of yellow lupin, Lupinus luteus L. cv. Topaz. Their structures were unambiguously determined to be 5, 7, 4'-trihydroxy-3-methoxy-6-(3, 3-dimethylallyl)flavone (1) and 5, 7, 4'-trihydroxy-6-(3-hydroxy-3-methylbutyl)-3-methoxyflavone (5) by a combination of chemical and spectroscopic methods, and the new flavones were named topazolin and topazolin hydrate, respectively.
Antifungal tests against the growth of Cladosporium herbarum indicated that, in spite of its phenolic nature and the possession of an isopentenyl sidechain, topazolin (1) had only weak fungitoxic activity.

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Further support to the hypothesis that antimutagenic activities of α, β-unsaturated carbonyl compounds against UV-induced mutagenesis of E. coli may be due to an interaction with thiol groups were obtained by an experiment with the supplement of glatathione to the assay medium. Antimutagenic activity against MNNG induced mutation was also observed.

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The protoplast fusion of Aspergillus sojae was studied to develop a breeding system for the more desirable koji-molds in the production of shoyu (Japanese fermented soy-sauce) with special attention as to their enzyme productivities. Double-marker mutants, as to conidial color and nutritional requirement, were first derived from the parental strains selected from genealogically unrelated A. sojae cultures showing characteristic enzyme-productivities. Protoplasts of the mutants were obtained in high yields by using a combined enzyme-system composed of the enzyme preparation obtained from a culture filtrate of Bacillus circulans IAM 1165 and commercial chitinase (ICN). The protoplasts were fused in PEG 6000. Many of the fused products that grew on minimal regeneration plates were found to be heterokaryons. Stable heterozygous diploids with green conidia were induced from heterokaryons on UV treatment. One hundred and thirty diploid strains were obtained and their enzyme-productivities were assayed. The activities of both protease and glutaminase were distributed within the range of those of the parents, and in most strains, the sum of the two activities did not exceed those in the cases of the parents. Exceptionally, a few diploids showing well-balanced high productivity of both enzymes were also obtained.

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Enzymes that catalyze the oxidation or reduction of methylglyoxal were screened for in several prokaryotic and eukaryotic microorganisms. Methylglyoxal-reducing activity was found to be widely distributed at considerably high levels in microorganisms. Methylglyoxal-oxidizing activity was detected only in cells of Pseudomonas putida among the organisms examined. The enzyme responsible for the methylglyoxal oxidation was purified approximately 240 fold from a cell extract of P. putida. The enzyme consisted of a single polypeptide chain with a molecular weight of 42, 000 and showed a pH optimum of 8.0. The enzyme was active on 2-ketoaldehydes [glyoxal, methylglyoxal (Km = 1.0 HIM) and phenylglyoxal] and some aldehydes (formaldehyde, acetaldehyde, glycolaldehyde and propionaldehyde) in the presence of NAD (Km = 0.1 nM). The bacterial methylglyoxal-oxidizing enzyme appeared similar to goat liver 2-ketoaldehyde dehydrogenase in molecular weight and structure, but was different in its substrate affinity.

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One molecular species of prothoracicotropic hormone with a molecular weight of about 22, 000 (22K-PTTH) of the silkworm, Bombyx mori, was isolated from 5 × 10⁵ adult heads. The purification procedure consisted of 16 steps including defatting, salt-extraction, fractional precipitations, conventional column chromatographies, and high performance liquid chromatographies. An approximately 5 × 10⁶-fold purification was attained to yield 5.4 μg (0.25 nmol) of the pure hormone with a recovery of 3.3%. Injection of 0.11 ng of the purified 22K-PTTH could elicit adult development in a brainless Bombyx pupa. 22K-PTTH is a basic protein (pI 7.7 - 8.7) containing disulfide bond(s). The amino-terminal amino acid sequence of 22K-PTTH was determined to be Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ile-Pro-Asp-Pro-.

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The activity of galactanase from Penicillium citrinum was enhanced by the addition of HgCl₂ and KC1 to the reaction mixture. The enhancement could be observed only when o-nitrophenyl-β-D-galactopyranoside (ONPG), p-nitrophenyl-β-D-galactopyranoside (PNPG), and β-1, 4-linked galactobiose were used as substrates, i.e., not when soy bean arabinogalactan and o-nitrophenyl galactobioside were used.
Among the metal ions tested, only HgCl₂ was effective. KCl could be replaced by KBr but not by KF or KI.
The extent of the activation depended on the concentrations of HgCl₂ and KCl, and the maximum activation (about 5.5-fold, as measured as the amount of o-nitrophenol liberated in a definite time) was observed when 0.1 IBM HgCl₂ and 25 mM KCl were added. The activity enhancement was also dependent on the concentration of ONPG. The activation was evident only when the concentration of the substrate was low and its extent decreased with an increase in the substrate concentration.

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The capturing interaction between an insoluble pyridinium-type polymer and bacterial cells was investigated. The strength of the interaction was evaluated by the removal coefficient reported previously based on the initial rate of decrease of viable cell counts caused by the presence of the polymer. Hydrophobic bacteria and hydrophilic bacteria showed distinct differences in the capturing interaction. With hydrophobic bacteria, electrostatic interaction as well as hydrophobic interaction between the polymer and cells appeared to be important. With hydrophilic bacteria, however, other factors such as solvent (water) mediated forces and hydrodynamic forces were suggested.

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A simple and selective assay method for a trypsin-sensitive cholecystokinin (CCK)-releasing peptide ('monitor peptide') found in rat bile-pancreatic juice is reported. This peptide also has trypsin inhibitory activity which could be used as an index for measurement, except that it was difficult to assay the inhibitory activity of the peptide directly in the bile-pancreatic juice because of abundant endogenous trypsin. The peptide and trypsin were purified from rat bile-pancreatic juice, and their sensitivities to heat treatment was examined. Trypsin(ogen) was completely eliminated by being heated at 80°C for 40min in the presence of 0.5 M NaCl, pH 2.5, whereas the trypsin inhibitory activity of the peptide was above 95% of the original level after this treatment. Using the difference, we established assay conditions for trypsin inhibition by the peptide in which the effects of endogenous trypsin were removed by heating. This assay method was then used to show that there was a close relationship between trypsin(ogen) and the peptide in the bile-pancreatic juice. We estimated that the trypsin inhibitory activity of the peptide masks 2-5% of the trypsin activity in the bile-pancreatic juice from Wistar male rats fed a stock diet. The optimum conditions for activation of trypsinogen and chymotrypsinogen in rat bile-pancreatic juice were also studied. At 37°C, trypsinogen and chymotrypsinogen were maximally activated by enterokinase 5% of protein being activated for 40 and 6 min, respectively.

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We have previously constructed hEGF secretion vector pTA1522 in which phoA signal peptide-hEGF hybrid gene was expressed under the control of the phoA promoter, and have shown that Escherichia coli carrying pTA1522 produced authentic hEGF in large amounts. The present report describes the relative efficacies of a number of secretion vectors that utilized various promoters (phoA, trp, rightward and leftward promoter of phage λ), signal peptides (phoA, bla), and antibotic resistance markers (Amp^r, Km^r) in different combinations. The most effective was pTA2532 with phoA promoter, a bla signal peptide coding region, and Km^r gene. The amount of hEGF secreted was 2.12×10⁵ molecules per cell.

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Purification of the precipitates obtained from the juice oil of Citrus hassaku by chromatography afforded 7-geranyloxycoumarin (aurapten) and two compounds (mp 43-45°C and 122-124°C), whose structures were determined to be epoxyaurapten and marmin on the basis of spectral evidence. These compounds were isolated from Citrus hassaku for the first time. The spasmolytic activity was tested of aurapten, epoxyaurapten, marmin and their related compounds, which were synthesized from aurapten and marmin, on the small intestine removed from a male guinea pig. Epoxyaurapten exhibited the highest activity among them against the small intestine's contraction induced by BaCl₂.

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The gene (gsh I) for γ-glutamylcysteine synthetase (GSH I) has an unusual translational initiation codon TTG. By means of a method involving a mismatched primer, this initiation codon, TTG, of the gsh I gene was replaced by the normal ATG. The GSH I activity increased 50% with the conversion. Expression of the fused genes (gsh I: lacZ) under the control of the gsh I initiation (ATG or TTG) was increased to the same level as above. Although the extent was not so remarkable as in the cases of β-galactosidase and adenylatecyclase, we deduced that the translational efficiency of the gsh I gene was affected by the base substitution of the initiation codon, TTG, to ATG.

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In the present work, an apoprotein solution was prepared from hen's egg yolk low density lipoprotein (LDL) without using detergents; LDL was delipidated with chloroform-methanol (2:1) and solubilized by sonication with 80% ethylene glycol. An apoLDL-phospholipid complex was prepared by sonicating this apoprotein solution with an egg yolk lecithin suspension. Although high-molecular-weight polypeptides of apoLDL formed an insoluble complex with lecithin, its lowmolecular-weight polypeptides formed a soluble complex. The soluble apoLDL-phospholipid complex gave only one peak on gel filtration on Sephacryl S-400.
Emulsifying properties of the soluble apoLDL-phospholipid complex were much bettej than those of either lecithin vesicles or lecithin suspensions with the same concentration of phospholipid, and almost the same as those of LDL. These results seem to show that the superior emulsifying properties of LDL depend on the characteristic structure of its lipid-protein complexes.

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A total synthesis of optically active pyriculol is described. The Wittig reaction between an aldehyde 19 and a triphenylphosphonium ylide 12 gave an intermediate 20. Successive treatment of 20 with p-toluenesulfonic acid, active manganese dioxide, and potassium carbonate gave (3'R, 4'S)-pyriculol (23), which was identical with natural pyriculol (1) in all respects. From this synthesis, the absolute stereochemistry of pyriculol (1) was determined to be 2-[(3'R, 4'S')-3/, 4'-dihydroxy(1'E, 5'E)-1', 5'-heptadienyl]-6-hydroxybenzaldehyde.

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A novel synthesis of (±)-methyl epijasomonate (2) and the first synthesis of (±)-methyl cucurbate (4) were achieved starting from 2-allylcyclohexane-1, 3-dione (8). The synthetic epimer 2 had a stronger jasmin flavor than the trans-isomer 1 with 95% purity.

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A mature human interleukin 2 (hIL-2) and its derivatives that lacked the N-terminal portion were expressed in Escherichia coli under the control of the phage λ P_L promoter. They accumulated in the form of insoluble inclusion bodies and accounted for about 30% of the total cellular protein. The mature hIL-2 and its derivatives were further purified and their in vitro biological activity was compared in an IL-2 microassay. The results suggested that the hIL-2 derivatives without the N-terminal three or five amino acids were as active as intact hIL-2 and that those without the N-terminal eight or nine amino acids were less active than the intact form.

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(±)-Methyl 1, 3-dimethyl-2-oxo-3-cyclohexene-1-carboxylate (7) was synthesized from methyl propionate via Claisen condensation followed by Robinson annelation with acrolein. Lithium aluminum hydride reduction and manganese dioxide oxidation of 7 gave (±)-6-hydroxymethyl-2, 6-dimethyl-2-cyclohexen-1-one (3), which is a useful new building block for natural product syntheses. Next, 3 was converted to (±)-8'-acetoxy-3'-oxo-β-ionone (16). A Wittig reaction of 16 with 3-(2-methyl-1, 3-dioxolan-2-yl)propylidenetriphenylphosphorane in tetrahydrofuran, with subsequent deprotection, afforded the fungal sexual pro hormone, (±)-(7E, 9Z)-trisporol B (1), and its (9E)-isomer (19).

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Five novel C₃₀ triterpenoids, ganoderic acids Mg (10), Mh (11), Mi (12), Mj (13) and Mk (14), were isolated from the mycelial mat of a G. lucidum strain, which produces C₂₇ lucidenic acids in the fruiting body. Their structures were determined by spectroscopic analysis and chemical conversion.

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A putative structural gene (RHA). coding for rhodotorucine A, a farnesyl peptide mating pheromone in cells of mating type A of Rhodosporidium toruloides, has been cloned using synthetic oligonucleotides corresponding to the amino acid sequence of rhodotorucine A. The RHA gene contains sequences for four tandem copies of the rhodotorucine A peptide, each followed by a spacer peptide of three or four amino acids. The spacer peptides are hypothesized to contain signals for proteolytic processing and farnesylation. The results of Southern blot analysis revealed that the RHA gene is specifically contained in the genome of A-type cells, and not in that of complementary a-type cells. The existence of an additional homologous gene in the genome of A-type cells is also suggested.

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