Four peroxidase components, named RP-2, 4, 6, and 7, were isolated from rice (Oryza saliva L.) green leaves. Isoelectric focusing indicated that each preparation was homogeneous. The molecular weights of RP-2, 4, 6, and 7 estimated by SDS-PAGE were 48, 000, 48, 000, 40, 000, and 39, 500, and their isoelectric points were 5.4, 8.1, 9.3, and 9.2, respectively. The activity of every preparation was maximum around pH 5.0. Antisera against these purified enzymes were raised in rabbits. Ouchterlony double diffusion tests with these antisera suggested that RP-6 and 7 were immunochemically identical and RP-2 and 4 were identical in parts and that RP-6 and 7 were quite different from RP-2 and 4. Analysis of the N-terminal amino acid sequences also showed that these peroxidase components were classified into two groups. The polymerase chain reaction showed that RP-2 and/or RP-4 contained an active central region, which is homologous to other plant peroxidases.

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The whole cellular proteins of various hiochi bacteria were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of the electrophoretic patterns, the hiochi bacteria were generally divided into four distinguishable groups, agreeing with the conventional groupings based on physiological and biochemical characters; homo- and heterofermentative true hiochi bacilli and homo- and hetero-fermentative hiochi lactobacilli. The SDS-PAGE patterns of the other lactobacilli were clearly different from that of the hiochi bacteria. The SDS-PAGE patterns of SDS-extracted protein from hiochi bacteria can be used as a simple and a useful tool for identification of the microorganisms causing putrefaction of sake.

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The enzymic peptide synthesis in an organic medium was investigated by using papain in the presence of PEG. The added PEG enhanced the enzyme activity of papain for synthesis of the peptide, Boc-Gly-Asp(OBzl)OBzl. The activity of papain in the PEG-added system was higher than that in a PEG-free system.

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High hydrostatic pressures of 100 MPa to 300 MPa were applied to isolated myofibrils prepared from rabbit skeletal muscle to investigate the pressure-induced degradation of myofibrillar structure in the muscle.
A marked loss of the regular structure was observed in the phase-contrast image of the isolated myofibrils pressurized at 150 MPa, with further progress of the rupture of structure with increasing pressure applied. When exposed to pressures of 200 MPa or higher, clumping of the crushed myofibrils was observed. Electron microscopic studies of the pressurized myofibrils showed that the loss of M-line materials, rupture of I-filament, and the loss of the structural continuity with the loss of Z-line progressed in the myofibrils with increasing pressure applied. A sigmoidal relationship was obtained between the degree of solubilization and the intensity of the pressure applied to the isolated myofibrils. The electrophoretic analysis indicated that the amount and the species of the protein released from the myofibrils at each stage of the pressurization corresponded to the disruption of the ultrastructure in the myofibrils.

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The mammalian genome congains long interspersed repetitive sequences, but the role of these repetitive sequence is not clear. A cDNA clone has been isolated that contains part of the LI sequences from a cDNA library of rat liver. The DNA sequence analysis showed the homology of cDNA to several reverse transcriptases. The homology between the amino acid sequences predicted from LI consensus sequences and reverse transcriptases has been reported previously. However, this is the first isolation of a cDNA clone containing a reverse transcriptase-like sequence.

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In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans API9-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mM and 1.5 mM NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mM NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-l, 5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mM, indicating that sulfite ions didn't inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe²⁺ - or S⁰-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans API9-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.

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A lectin was isolated from the fruiting bodies of Pholiota aurivella by preparative PAGE and named PAA (Pholiota aurivella agglutinin). PAA was a polymeric protein of more than several hundred kDa consisting of 18-kDa subunits. The amino acids were sequenced from the N-terminus of the lectin; they were YSVTTPNSVKGGTNQG. PAA agglutinated human erythrocytes regardless of blood type equally and Pronase treatment of erythrocytes increased the sensitivity to agglutination by the lectin. In a hemaggluting inhibition assay, none of the mono- and oligosaccharides used affected agglutination by the lectin. Among glycoproteins, asialofetuin was the best inhibitor. This is first isolated and characterized lectin from the family Strophariaceae.

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Gibberellin A₁ (GA₁), GA₃, GA₅₅ and 3-epi-GA₁ were identified by capillary gas chromatography/mass spectrometry (GC/MS) in young fruits of Carica papaya L. Gibberellin A₃ was dominant in young developing fruits, while GA₅₅ was the major component in abscising fruits. Furthermore, abscising fruits contained a neutral GA₃-conjugate.

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We established an efficient system for a high-level production of foreign gene products in Escherichia coli using a trans-activated gene expression under the control of a phage T4 regulatory signal cloned in a plasmid by T4 phage infection. The transcriptional and translational signal of the T4 uvsY gene cloned in a plasmid was fused translationally with the coding region of the lacZ gene. When E. coli cells carrying the uvsY-lacZ plasmid were infected with cytosine-substituting T4 phage at a multiplicity of infection of 5, the amount of β-galactosidase increased about 2-fold (tranis-activation) over that without phage infection. We examined conditions for the high-level production of a trans-activated gene product. We found that a large number of T4-infected cells in a lysis-inhibition state could be obtained by a low multiplicity of infection with cytosine-substituting T4 phage. Thus it is now possible to attain a high yield of the trans-activated gene products. We discuss the advantage of the trans-activated T4 uvsY regulatory signal for production of foreign products.

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A novel expression system was developed for the high level production of a labile protein in Escherichia coli. The regulatory signal of bacteriophage T4 uvsY gene was fused in frame with the coding region of human ventricular myosin alkali light chain (VLC1) gene. Expression from the regulatory signal was enhanced and continued in a lysis-inhibition state by infection with a cytosine-substituting T4 phage mutant. VLC1 protein was produced at a low level without infection because of its instability in the cells. Although the productivity was partly improved in a lon-deficient mutant without infection, it was improved about 100-fold with T4 phage infection. T4 phage produces protease inhibitor(s) (pin gene product) against proteases of host cell including the lon gene product (protease La).

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The interaction of bovine and human whey proteins with retinol and palmitic acid has been studied. Using gel filtration it was found that bovine β-lactoglobulin and α-lactalbumin and serum albumin from both species bind retinol in vitro while the ability to bind palmitic acid is restricted to bovine β-lactoglobulin and bovine and human serum albumin. Using equilibrium dialysis, β-lactoglobulin was found to display two binding sites for retinol per dimeric molecule with an association constant of 1.5×10⁴M^-1. Competition experiments showed that when the concentration ratio between total fatty acids and retinol is similar to that found in milk, palmitic acid competes with the binding of retinol to β-lactoglobulin.

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Twelve isomers of chemically unmodified tetradecenyl acetate were analyzed by two kinds of quadrupole mass spectrometers and a single focusing magnetic one, both of which are coupled with a gas chromatograph, for the identification of the double bond position. The spectra from one of the quadrupole instruments were interpreted in terms of mass spectral patterns on a fuzzy classification, in which the relative intensity ratios of five diagnostic pairs of the predominant ions were prefered in devising similarity indices. The method was tested with mass spectra from both a single focusing and another quadrupole instrument, and proved to provide all information necessary for identification of double bond position.

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Ventral eversible glands (VEG) were removed from larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), and extracted with methylene chloride. GC analysis of the VEG extract showed that the volatile components were about 98% saturated hydrocarbons. The distribution of individual hydrocarbons was as follows: n-tridecane, 0.1%; n-tetradecane, 2.4%; n-pentadecane, 96.2%; n-hexadecane, 0.3%; and n-heptadecane, 1.0%. Other components present at low levels were 1-pentadecene and three sesquiterpenes. A logarithmic increase in n-pentadecane level in the VEG occurred with successive molts from the third through fifth instar. The level of n-pentadecane in the VEG was empirically higher in fifth instar larvae feeding on corn foliage than in larvae feeding on a pinto bean diet or on cotton or peanut foliage.

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An agarase (agarose 4-glycanohydrolase, EC 3.2.1.81) was purified from the culture fluid of Pseudomonas sp. PT-5 by ammonium sulfate precipitation followed by Cellulofine GC-700m, Hydroxyapatite, Butyl-Toyopearl 650M, and Toyopearl-HW 508 column chromatography. The purified enzyme gave a single band on polyacrylamide gel disc electrophoresis and its molecular weight was 31, 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 3.6. The amino-terminal sequence was H•Ala-Asp-Trp-Asp-Gly-Leu-Ala-Val-Pro-Ala-Asp-Ala-Gly-Asp-Gly-. The enzyme was stable from pH 6 to 9 and had its maximum activity at pH 8.5. The enzyme rapidly reduced the viscosity of agarose solution and its activity was greatly inhibited by metal ions such as Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, and Al³⁺ at 1 mM concentration. The enzyme activity was elevated by 75% in the presence of 0.1 M NaCl.

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2-Aminothiazoline derivatives were synthesized by both hydrochloric acid-catalyzed cyclization of thiourea and by cyclization of β-aminoalkyl hydrogen sulfate with isothiocyanate in the presence of sodium hydroxide. Substituted 2-mercaptothiazoline derivatives were prepared by alkylation or acylation of the sodium salt of 2-mercaptothiazoline, which was obtained from β-aminoalkyl hydrogen sulfate with carbon disulfide. 2-(4-Chloro-0-toluidino)-2-thiazoline (111-16) was 33% as effective as octopamine at 100μM in stimulating adenylate cyclase of Penplaneta amencana ventral-nerve-cord homogenates. Its activity was nonadditive to the activity of octopamine. Stimulation of nerve-cord adenylate-cyclase activity by III-16 was inhibited by several antagonists, including mianserin, cyproheptadine, ehlorpromazine and gramine. The rank-order ability of these antagonists to block the activation by III-16 was identical to the rank-order ability of the same antagonists to block enzyme activation by octopamine. The β-adrenergic antagonist propranolol was less potent. These data suggest that 111-16 is a potent and selective agonist of octopamine-activated adenylate cyclase. Aminothiazolines which activated adenylate cyclase by 10-87% relative to octopamine also had acaricidal activity at 300 ppm, indicating a correlation between the in vitro octopaminergic-agonist activity and in vivo acaricidal activity of aminothiazolines.

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In the presence of 0.5 mM EGTA (a calcium chelator), the muscarinic agonist carbachol at 0.1 mM inhibited octopamine-stimulated cyclic AMP (cAMP) production in intact ventral nerve cords of Periplaneta americana. This effect was reduced by the addition of 0.1 mM atropine (a muscarinic antagonist), suggesting that the effect is mediated via muscarinic acetylcholine receptors (mAChR). This inhibitory effect of carbachol was not observed in the synaptosomes of nerve cords, suggesting that the inhibitory effect was not due to direct coupling of the mAChR to adenylate cyclase but mediated by other second messengers. The inhibitory effect seems to be due to an increase in the intracellular calcium level. In the presence of exogeneous extracellular 0.25 mM Ca²⁺ the octopamine-stimulated cAMP production was lower than that in the absence of extracellular Ca²⁺, and the cAMP levels were not further reduced by adding carbachol to the octopamine-stimulated cAMP production system. Washing did not remove all of the inhibitory activity of carbachol in intact nerve cords, indicating that carbachol modulated octopaminergic neurotransmission irreversibly.

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An aryl-β-cellobioside-hydrolyzing enzyme was obtained from Escherichia coli WL66 infected with a recombinant phage, λCG23, harboring a 7.2-Kbp DNA fragment from Cellvibrio gilvus. It was purified by hydrophobic, ion exchange and gel filtration chromatographies to an electrophoretically homogeneous state. The purified enzyme has a molecular weight of 82, 000 and a pI of 6.0, and its amino terminal amino acid sequence was identified. It produced glucose from a series of cellooligosaccharides and aryl-β-glucosides such as 4-methylumbelliferyl-β-glucoside and pnitrophenyl-β-glucoside. Although the enzyme released 4-methyumbelliferon from 4-methylumbelliferyl-β-cellobioside, the rate of its release was much slower than the rate of glucose release. From these results the purified enzyme was identified as β-glucosidase (EC 3.2.1.21). However, its action on cellobiose was substantially lower than the action on higher cellooligosaccharides.

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Utilizing the carbon framework (C3-C6) and chirality (at C4 and C5) of D-glucose, (2S, 3S, 4E and 2R, 3S, 4E)-2-amino-4-octadeceiie-1, 3-diol (sphingosine analogues) and four Stereoisomers (2S, 3S, 4E-, 2S, 3S, 4Z-, 2R, 3S, 4E-, and 2R, 3S, 4Z-) of the ceramide derivatives were synthesized in a stereocontrolled way.

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Cold-pressed oil (CPO) components from 8 varieties of pummelos were identified and measured. γ-Terpinene was the second major component in 4 varieties, but only a minor component in the others. Cadina-1(10), 6, 8-triene was identified by GC-MS in addition to the 63 compounds identified already.
Multivariate analyses were applied to 37 kinds of citrus fruits including pummelos and other species. The pummelo was classified into 2 groups by cluster analysis, and into 3 groups by principal component analysis on the basis of the oxygenated composition (w/w%) in fresh CPOs. The tendency for classification agreed in the two analyses. Nootkatone was the only discriminating component of the pummelo species from others. The results are also discussed in citrus species other than pummelo, being compared with the botanical classifications.

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Carboxylesterase from Ochrobactrum anthropi was purified to homogeneity by a combination of DEAE-cellulose, Sephadex G-100, and bydroxyapatite column Chromatographies. The molecular weight of the enzyme was calculated to be 58, 000 by Sephadex G-75 gel filtration. SDS-polyacrylamide gel electrophoresis showed that the enzyme consisted of two identical subunits with a molecular weight of 30, 000. The enzyme was inactivated by several organophosphates and sulfhydryl reagents. The esterase hydrolyzed only acetate and propionate esters. In the presence of Triton X-100, the esterase hydrolyzed l-menthyl acetate but not its optical isomer. This result suggests that the enzyme can be used for the optical resolution of l-menthol.

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We have proposed 21 method to produce an oligopeptide mixture from zein by enzymatic modification to improve the Fischer ratio. In this study we produced the oligopeptide mixture on an enlarged scale and evaluated it in comparison with a corresponding amino acid mixture by feeding tests with normal and liver-injured rats. Both of the oligopeptide mixture and the amino acid mixture enhanced the Fischer ratio in plasma and brain at a similar degree. In rats suffering from severe hepatic injury, undesirable aromatic amino acids were absorbed from the oligopeptide mixture to a less extent than from the amino acid mixture. Peptide feeding, as well as amino acid feeding, resulted in decreasing concentrations of tryptophan and serotonin and in keeping the concentrations of dopamine and norepinephrine in cerebrum. These results suggest that the oligopeptide mixture is useful for patients with severe heptic disease.

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The nutritional efficiency of nicotinamide N-oxide and N'-methylmcotinamide as substitutes for nicotinic acid was investigated using weanling rats fed with a nicotinic acid-free and tryptophan-limited diet (basal diet; negative control), the basal diet containing 0.003% nicotinic acid (positive control), or basal diet containing various amounts of nicotinamide N-oxide or N'-methylnicotinamide. The relative niacin activity of nicotinamide N-oxide and N'-methylnicotinamide to nicotinic acid was about 1/5 and 1/2 on a weight basis, respectively, judged from the following comparisons: body weight gain, food intake, food efficiency ratio, the NAD contents in liver and blood, the total nicotinamide content in liver, and the urinary excretion of nicotinamide and its metabolites such as N¹-methylnicotinamide, N¹ -methyl-2-pyridone-5-carboxamide, and N¹ -methyl-4-pyridone-3-carboxamide.

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An α-L-arabinosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) that can degrade both purified water soluble polysaccharide from soybean seeds and p-nitrophenyl-α-L-arabinofuranoside was purified 453-fold to near homogeneity from cotyledons of 4-day-old soybean seedlings.
The optimum pH and K_m against p-nitrophenyl-α-L-arabinofuranoside of the purified enzyme were 4.8 and 0.53 mM, respectively. The enzyme was activated by Ca²⁺ and Zn²⁺ ions, but Cu²⁺, Hg²⁺, EDTA, and L-arabonic-γ-lactone inhibited the enzyme activity. The molecular weight of the enzyme subunit was estimated to be 87, 000 by SDS-PAGE.

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Daily changes of nutritional status during total parenteral nutrition (TPN) were studied in partially hepatectomized (PH) rats. When PH rats were parenterally infused with 8 to 10 g of amino acids and 40 to 60 g of glucose per kg daily for 1 to 3 days, the nitrogen balances in PH rats were positive during the infusion period, but the balance was significantly lower on day 3 than that in nonhepatectomized (NPH) rats. Urinary total catecholamine levels in PH rats tended to be higher (45 to 70%) than those in NPH rats. Throughout the infusion period, plasma Met and Phe levels were elevated, and plasma albumin and total protein levels were decreased in PH rats as compared with NPH rats. Marked increases of plasma GOT and GPT activities were observed in PH rats on day 1 and the elevated activities were gradually normalized during TPN. These results indicate that the nutritional status of PH rats was inferior to that of NPH rats under the TPN conditions but that TPN was rapidly restoring the nutritional status of PH rats.

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Starting from milbemycin D (1), milbemycin A₄ (2) and milbemycin A₃ (3), a series of 5-keto-5-oxime derivatives were synthesized by selective oximation at the α, β-conjugated carbonyl function of the 5-ketomilbemycins (4-6). The activities of the synthesized compounds were studied in dogs naturally infested with microfilariae of Dirofilaria immitis. The 5-keto-5-oximes of milbemycin D (7), A₄ (8) and A₃ (9) had quite high efficacy to control the microfilariae and more potency than their parents, while the 5-O-acyl oximes (11-15) also exhibited high activity.

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