The effects of okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, from the sponge Halichondria okadai Kadota, on the embryonic development of the starfish Asterina pectinifera, were investigated. When cultured in okadaic acid from fertilization, the embryo divided synchronously without any abnormalities such as lysis, swelling or morphological changes different from control embryos up to the 32-cell stage. However, okadaic acid prevented development before the onset of blastulation. Cytological examination showed that chromosomes condensed but did not align in a plane in the mitotic apparatus in each of the blastomeres of the treated embryo at the sixth cleavage, suggesting that this was the root cause of the arrest of further development.

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For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E.coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets in a glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.

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Biotinylation of fusion proteins in E.coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences : one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine(Bct) to the carboxyl terminal ; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of β-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.

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Polyphenol oxidase was extracted from banana buds in the presences of Triton X-100, isoascorbate, and Polyclar AT, and two isozymes I and II have been separated and partially purified by chromatographies on Butyl Toyopearl 650 and DEAE-cellulose. I and II had different mobility in polyacrylamide gel electrophoresis with optimum pHs of 6.8 and 5.5, respectively. Both enzymes showed the apparent K_m values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCl with the K_i values of 140 mM and 40 mM, respectively. I and II have a high heat stability, and 88 and 95% of the initial activities were retained after 1-hr incubation at 70°C, respectively.

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Poly(γ-glutamic acid)(PGA) production in Bacillus subtilis IFO3335 was studied. When citric acid as a carbon source was added to a glutamic acid medium containing L-glutamic acid and ammonium sulfate, a large amount of pure PGA was produced. On the other hand, when glucose was added to the glutamic acid medium, a by-product was produced, which seemed to be a polysaccharide. Moreover, the mode of hydrolysis was investigated with PGA in aqueous solutions at 80, 100, and 120°C by monitoring the time-dependent changes in the molecular weights. Hydrolytic degradation of PGA was found to proceed through a random chain scission.

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Protein bodies in embryonic axes of soybean seeds have inclusion structures containing phytin globoids. Biogenesis of the protein bodies during seed development was examined by transmission electron microscopy. Protein bodies in embryonic axes originated from central vacuoles. The central vacuole in embryonic axes subdivided into smaller vacuoles with internal membranous structure. Then the subdivided vacuoles were directly associated with rough endoplasmic reticulum (rER), and were filled with proteinaceous matrix from the peripheral region. The increase of matrix was simultaneous with accumulation of β-conglycinin estimated by SDS-polyacrylamide gel electrophoresis. Glycinin-rich granules that had been found in developing cotyledons were not observed in embryonic axes. After proteinaceous matrix filled the protein bodies, electron-transparent regions presumably surrounded by a single membrane appeared in the matrix. Phytin globoids were constructed in this internal structures of protein bodies as the final step of protein body formation.

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HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK₁ cells. At concentrations from 0.1 to 100 μg/ml (=2.5×10^-8-2.5×10^-5M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10^-8M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10^-5M sodium nitroprusside. HS-142-1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulatiion of cyclic GMP in LLC-PK₁ cells through interacting with guanylyl cyclase-linked receptors.

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Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and /or on the growth of dwarf rice (Oryza sativa L.cv.Tan-ginbozu) and cucumber (Cucumis sativus L.cv.Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ inhibited the conversion of [2, 3-³H₂]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P.vulgaris and C.maxima at 3μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P.vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C.maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higer. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. IN a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.

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An enzymic-HPLC method was successfully applied to the simultaneous analysis of the galacturonate and neutral sugar contents of pectic substances. A mixture of seven neutral sugars that are present in oridnary pectins was eluted as one peak on a Shodex SUGAR SH1821 column, using 0.001N sulfuric acid as the eluent ; the peak was completely separated from that of galacturonate. No difference in the peak-area ratios of individual neutral sugars to glycerol (internal standard) was found among the seven ; the relationship between the peak response and concentration was strictly linear throughout the entire concentration range studied. Seventeen pectic samples, including pectins, pectates, and rhamnogalacturonan fragments, were completely prehydrolyzed by Driselase, an industrial enzyme product from Irpex lacteus, and then analyzed for their constituent sugar contents. The enzymic-HPLC method was simple, accurate, and particularly useful for routine pectin analyses.

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Fermentation of waste fish treated with Aspergillus oryzae, Aspergillus sojae K, and Saccharomyces cerevisiae IFO 2114 were studied independently and combined. Three microorganisms decreased the POV, MDA, and COV of fish meal at different rates. The optimum conditions for fermentation with the combination of three microorganisms was found at 30°C for 20-hr fermentation. Almost no difference was observed in the chemical composition or amino acid spectra of the protein hydrolysates of the fermented and nonfermented fish meal. On the quantity of water-soluble amino acids, the highest increase was in glutamate among others, but histidine was decreased by the combination of the three microorganisms. With A. oryzae, the highest increase was in phenylalanine and with A. sojae K in threonine. A great change was observed in some fatty acids content. Myristic acid (14 : 0) was decreased while the highest increase occurred in the linoleic acid (18 : 2) content by fermentation with the combination of three microorganisms. The same phenomenum was observed with A. oryzae and A. sojae K.S. cerevisiae has a weaker effect than A.oryzae and A. sojae K in fermentation of waste fish.

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2-(Substituted benzylamino)-2-thiazolines (SBAT) were synthesized by a hydrochloric acid-catalyzed cyclization of the corresponding thioureas, using a reaction of 2-methylthio-2-thiazoline with substituted benzylamines or by alkylating 2-amino-2-thiazoline. 2-(Alkylthio)-2-thiazolines were obtained by alkylating 2-mercaptothiazoline. Most of the SBAT compounds activated adenylate cyclase in homogenates of cockroach ventral nerve cords ; the effect of introducing substituents at the phenyl of the SBAT compounds on octopaminergic agonist activity was not significant. 2-[β-(Substituted phenyl)ethylamino]-2-thiazolines and 2-(alkylthio)-2-thiazolines were not significant octopaminergic agonists. Washing removed nearly all of the activity of one of the SBAT compounds, suggesting that the SBAT compounds bound reversibly to the octopaminergic receptor.

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The activities of microbial lipases were inhibited by bile salts in a non-emulsifying assay system. To protect lipase activities from inactivation, the effects of proteins and amino acids were investigated. Bovine serum albumin (BSA) and α-lactalbumin (α-LA) stored the bile salts inhibited microbial lipases. Among N-end amino groups contained in BSA, L-histidine restored the activities of the bile salts inhibited microbial lipases. On the other hand, pancreatic lipase activity was stimulated by not only BSA, but L-histidine and L-aspartic acid as N-end amino groups of BSA and additionally accelerated it in combination with bile salts.

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A novel enzymatic process was developed to simultaneously produce high-maltose syrup and high-protein rice flour from milled rice. This process would greatly increase the commercial value of rice. A slurry of rice flour was first liquefied with α-amylase and then centrifuged, the precipitated fraction being recovered as high-protein rice flour. The supernatant fraction was then saccharified with β-amylase and debranching enzymes (isoamylase and/or pullulanase) together to produce high-maltose syrup under various conditions. The yield of high-maltose syrup was affected by the temperature, pH, DE value of the liquefied starch, the enzyme combination and variety of rice. As and example, 0.64g of high-maltose syroup (83% maltose and 2.6% glucose) and 0.36g of high-protein rice flour (29% protein, 39% starch and 14% sugar) could be produced from one gram of dry rice (Taichung Native 1).

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An enzyme which catalyzes the NADPH-dependent reduction of 3-deoxyglucosone (3DG) was isolated and purified from porcine liver by ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, Sephadex G-100, Blue Sepharose CL-6B, and Sephadex G-100 column chromatographies. The pH for optimum enzyme activity was 6.5. 3DG was a good substrate, and glucose and fructose could also be reduced at a significant rate. The K_m values for 3DG, methylglyoxal and glyceraldehyde were 2.5, 1.9, and 4.9mM, respectively. The molecular mass of the enzyme was estimated to be 67, 000, and the enzyme is proposed to be a dimer composed of identical subunits. The activity of the enzyme was completely inhibited by p-chloromercuribenzoate, and the enzyme is estimated to be an aldose reductase.

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Tandem mass spectrometry with a four-sector type mass spectrometer was used to elucidate the structures of minor components of the peptidyl antibiotic P168s(leucinostatins). As N-terminal fragments, ions by B-type cleavage were dominant, while V-type cleavages were observed along with X, Y, and Z types as C-terminal ions. The V-type ions were predominant in the cleavages of the amino terminals of leucyl and hydroxyleucyl residues. The structures of several minor components could be deduced from the tandem mass spectra.

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Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.

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A procedure for obtaining pure glycoasparagine specimens was developed. This method constitutes ion-exchange chromatography in a DEAE-Sephadex A-25 (borate form) column, using a linear gradient of borate buffer as the eluent. With no conversion, almost all the individual glycoasparagines, including some new ones, derived from ovalbumin could be isolated in pure form. By the borate-ion exchange chromatographic technique presented here, different types of neutral glycoasparagnes having a differing ability to form a complex with borate ions.

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Four novel antifungal compounds, gamahonolides A and B, gamahorin, and 5-hydroxy-4-phenyl-2(5H)-furanone, were isolated from stromata of Epichloe typhina on Phleum pratense. Their structures were determined by spectroscopic methods. The absolute configuration of gamahonolide A was determined by its ORD spectrum and ¹H-NMR shift difference between the diastereomeric pair of its O-methylmandelates. The stereochemistry of gamahorin was determined by NOE difference spectra and its CD spectrum.

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Dietary fibers, alginate and defatted corn fiber, sorbed food mutagens, Trp-P-1 and Glu-P-1, which are heterocyclic amines formed in the cooking process. The sorption behavior of the heterocyclic amines to defatted corn fiber and alginates was analyzed under pH-controlled conditions. Glu-P-1 and alginic acid had pK_a values of 4.2 and 3.6, respectively, whereas Trp-P-1, which showed alkaline in solution, possessed two pK_a values of 4.8 and 7.7. Defatted corn fiber, which was mainly composed of cellulose and hemicellulose, did not show a significant pK_a value. The amount of sorbed Trp-P-1 to the alginates increased as the pH value of the buffer was elevated, and was much more than that sorbed to defatted corn fiber at each pH condition. These results suggest that the alginates held the amino group of Trp-P-1 or Glu-P-1 on their carboxyl group as a cation exchanger.

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The effects of dietary fat and protein levels on the conversion of Trp-Nam were investigated. In rats fed with 20% casein diets, the Trp-Nam conversion ratio [(urinary excretion of Nam+MNA+2-Py+4-Py in μmol/day)/(daily Trp intake during urine collection in μmol/day)×100] was about 4.3% for the groups fed with the 20% corn oil and 20% soybean oil diets, 2.8% for the group fed with the 20% lard diet, and 2.1% for the group fed with the no fat diet. In rats fed with 40% casein diets, a similar phenomenon was observed, but the ratios were 2.0%, 2.4%, 1.6%, and 0.8% for the groups fed with the 20% corn oil, 20% soybean oil, 20% lard, and no fat diets, respectively. From these results, it was found that an increase in fat intake elevated the conversion ratio regardless of the dietary protein level, while an increase in protein intake reduced it.

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This paper describes the glycation of myofibrillar proteins and its relationship with aging. The identification of N^ε-fructoselysine in myofibrillar protein was done by the HPLC procedure and mass spectrometry. The N^ε-fructoselysine content in mouse myofibrils during the experimental period of 59 weeks rose slightly from 0.56 to 0.74 nmol/mg of protein. That in mouse actomyosin was higher and increased exponentially from 2.32 to 4.98 nmol/mg of protein during a period of 27 weeks, indicating a relationship between the glycation of mouse actomyosin and age. In the case of rats, although the N^ε-fructoselysine content of myofibrillar proteins did not show a clear change like that of mice, the content in actomyosin gradually increased with age. Taking into account the decrease in muscle protein turnover with age that has been described in previous papers, these results imply a relationship between the glycation of myofibrillar protein and age. This is the first report on the glycation of muscle protein.

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Genomes of many eubacterial strains have been shown to encode for multiple rpoD-related genes. In this report, we describe the identification of the multiple rpoD-related genes of cyanobacterial strains. DNAs of three cyanobacterial strains, Anabaena sp.PCC7120, Synechococcus sp.PCC7942, and Synechocystis sp. PCC6803, were examined by Southern hybridization, using a synthetic probe designed for detecting rpoD or rpoD-related genes. Four or five hybridization signals were found in each DNA. Four DNA regions of Synechococcus sp.PCC7942 corresponding to the hybridization signals were cloned and partially sequenced. The sequence data indicate the presence of genes, named rpoD1, rpoD2, rpoD3, and rpoD4, whose products are highly similar to the basic structure of the principal σ factors of eubacterial strains. The rpoD1 gene showed the greatest similarity to the sigA gene of Anabaena sp.PCC7120.

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A microbial lipase from Pseudomonas aeruginosa TE3285 was treated in anhydrous diisopropyl ether with three kinds of serine-reactive reagents, ethyl p-nitrophenyl methylphosphonate (ENMP), diisopropyl fluorophosphate (DFP), and phenylmethylsulfonyl fluoride (PMSF) to lose its catalytic activity for both transesterification in an organic solvent and ester hydrolysis in aqueous system. In contrast with the facile inactivation in an organic solvent, no or very slow inactivation was observed in an aqueous solution. The lipase was shown to behave more like a typical serine enzyme in an organic solvent than in aqueous solution with regard to the chemical inactivation by serine-reactive reagents. The unique behavior of the lipase in an organic solvent may be associated with inferfacial activation of the lipase, which is one of the most distinct characteristics of the lipase family, and the activiation of lipase could be induced by a hydrophobic interaction with an organic solvent.

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Grayanotoxin loosely binds to the neurotoxin receptor site 2 on Na channels. 14R-N-[4-(1-Azi-2, 2, 2-trifluoroethyl)]benzyliminograyanotoxane-3β, 5, 6β, 16-tetraol (4) and 17-N-[4-(1-Azi-2, 2, 2-trifluoroethyl)]-benzyliminograyanotox-15-ene-3β, 5, 6β, 14R-tetraol (5) were synthesized and applied intracellularly to squid axons. On UV irradiation, their membrane potential produced by a dose of 4 or 5 rose to higher level than that in the dark. However, in the case of 5, even when the axons were internally perfused with toxin-free solution after the dose, the potential level did not fall in contrast to 4. It suggested that 5 covalently bound to the receptor involved in the gating mechanism of Na channels.

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