Our previous findings concerning the lipoxygenase (LOX) activity increment in infected rice plants and anti-fungus activity of resulting oxygenated fatty acids have prompted us to examine whether LOX activity increases in higher plants in response to infection with pathogens peculiar to individual plants. This study found that LOX activity increases in several kinds of infected higher plants, particularly in infected tomato leaves. Linolenic acid is oxidized to 9S-hydroperoxy-10E 12Z, 15Z-octadecatrienoic acid by the action of tomato LOX. This finding provides another example concerning the LOX activity increment in infected higher plant. The oxidation products had antimicrobial activity toward several kinds of pathogens.

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Overexpression of the asnA gene from Escherichia coli K-12 coding for asparagine synthetase (EC 6.3.1.1) was achieved with a plasmid, pUNAd37, a derivative of pUC18, in E. coli. The plasmid was constructed by optimizing a DNA sequence between the promoter and the ribosome binding region. The enzyme, comprising ca. 15% of the total soluble protein in the E. coli cell, was readily purified to apparent homogeneity by DEAE-Cellulofine and Blue-Cellulofine column chromatographies. The aminoterminal sequence, amino acid composition, and molecular weight of the purified protein agreed with the predicted values based on the DNA sequence of the gene. Furthermore the native molecular weight measured by gel filtration confirmed that asparagine synthetase exists as a dimer of identical subunits.

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The mode of action of the endo α-1, 4 polygalactosaminidase from Pseudomonas sp. 881 on galactosaminooligosaccharides (GOSs) was studied. The enzyme could hydrolyze α-1, 4 polygalactosamine to GOSs by the endo-split manner. Tetraose and longer GOSs were hydrolyzed to galactosaminobiose and galactosaminotriose as the final products. Galactosaminomonomer (galactosamine) could not be produced as an enzymatic product. From the dependency of kinetic parameters on the chain lengths of the substrates, it was suggested that the enzyme has 8 subsites. A catalytic site of the enzyme is located between the third and the fourth sites from the non-reducing end, since the main product from GOSs was galactosaminotriose, and galactosaminotetraitol remained in the hydrolyzate of galactosaminoheptaitol digestion. The enzyme showed transglycosylating activity on GOS4.

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The level of the inclusion complex between β-cyclodextrin (β-CD) and d-limonene in a pure β-CD and β-CD/maltodextrin mixed powder was measured by X-ray diffraction. The relative integral intensity at 2θ=5-7° and 11-12° correlated well with the molar fraction of d-limonene to the total moles of β-CD and the maltodextrin mixture. Solid state ¹³C-CP/MAS NMR results correlated well with those by X-ray diffraction.

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Two types of β-glucan synthases, GS-I and GS-II, were found in cultured rice cells (Oryza sativa L.). In glycerol density gradient centrifugation, GS-I activity peak co-migrated with a marker enzyme of the Golgi membrane, while GS-II co-migrated with the plasma membrane. Analysis of the reaction products of GS-I and GS-II, suggested that GS-I and GS-II were mainly β-1, 4, - and β-1, 3-glucosyltransferases, respectively. GS-I had a higher substrate affinity for UDP-glucose than GS-II, and needed divalent cations for its activity. Effects of nucleotides on the activity were also considerably different between GS-I and GS-II. GS-I was solubilized well with CHAPS and digitonin, and GS-II was solubilized effectively with sucrose monolaurate.

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The effects of NaCl concentration on bleomycin-induced cleavages of single-strand and double-strand DNA fragments containing the phage G4 origin of complementary DNA strand synthesis were investigated. It was found that bleomycin could be used as a reagent to analyze secondary and tertiary structures and subtle changes of DNA structures. The effects of NaCl concentration on cleavages of single-stranded DNA were distinct at every target site, indicating that the diversity of topolotical properties of DNA might change the selectivity of the bleomycin-induced DNA cleavage. These results showed alternative secondary structures within and close to the G4 origin of complementary DNA strand synthesis.

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For the classical solution synthesis of human epidermal growth factor (h-EGF), five protected peptide derivatives, Boc-Leu-Asp(OcHex)-Lys(Cl-Z)-Tyr(Br-Z)-Ala-OH (5), Boc-Val-Cys(MeBzl)-Met-Tyr(Br-Z)-Ile-Glu(OcHex)-Ala-OH (12), Boc-Tyr(Br-Z)-Cys(MeBzl)-Leu-His-Asp(OcHex)-Gly-OH (18), Boc-Cys(MeBzl)-Pro-Leu-Ser(Bzl)-His-Asp(OcHex)-Gly-OH (23) and Boc-Asn-Ser(Bzl)-Asp(OcHex)-Ser(Bzl)-Glu(OcHex)-OH (28) were synthesized to build up the sequence corresponding to 1-30.

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Human epidermal growth factor (h-EGF) composed of 53 amino acids bearing three intramolecular disulfide bridges was synthesized by the maximum protecting solution method. The synthetic h-EGF coincided with recombinant h-EGF by reverse-phase HPLC, and the sites of three intramolecular disulfide bridges were ascertained by a thermolytic digestion. The synthetic h-EGF possessed m/z 6215.7 in its FAB-MS as expected, and exhibited compatible mitogenic activity.

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Reaction rate constants between superoxide (O^-₂ ) and natural anti-oxidants, amino acids and some related sulfur-containing compounds were determined by quenching the chemiluminescence of a Cypridina luciferin analogue, 2-methyl-6-phenyl-3, 7-dihydroimidazo[1, 2-a]pyrazin-3-one (CLA) in pH 7.0 buffer solutions at 25°C as described in our previous report. The results are discussed in comparison with the literature data. We found that the present method can be applied to measured antioxidative activity of even a fairly unstable sample.

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The influence of the 6-methoxy-2-methoxymethyl-3-(3, 4-methylenedioxyphenyl)-1, 4-benzodioxan-7-yl group on the insecticidal activity of haedoxans was studied by synthesizing an analog without the (methoxymethyl)methinoxy moiety of the benzodioxanyl group to test for its activity on the housefly. The inactivity of the analog and its satellite compounds implies that the (methoxymethyl)methinoxy moiety is essential for the biological activity of haedoxans.

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Ten haedoxan analogs with the bond split between 2C and 3C of the 6-methoxy-2-methoxymethyl-3-(3, 4-methylenedioxy)phenyl-1, 4-benzodioxan-7-yl group of haedoxans were synthesized, and their insecticidal activity was assessed on the housefiy. The inactivity of an analog having a 2-methoxy-5-(2-methoxyethoxy)-4-(3, 4-methylenedioxybenzyloxy)phenyl instead of the 1, 4-benzodioxan-7-yl group made it evident that the benzodioxane framework is essential for the activity of haedoxans.

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The toxicity of citrinin heated at various temperatures was examined with HeLa cell and judged from the cell proliferation. Citrinin or heated citrinin (0.1, 1.0 or 2.5μg) was added to 3×10³ cells/100μl of medium, and the cell proliferation was monitored for 72hr. Lethal toxicity was clearly indicated by the addition of 2.5μg of citrinin, while the addition of 0.1 or 1.0μg of citrinin only slightly repressed the cell proliferation. Heating above 160°C was required to detoxify under dry conditions, whereas heating at 100°C reduced toxicity to some extent under aqueous conditions. Consequently, the lower the moisture, the higher was the temperature needed to detoxify citrinin. However, under the aqueous conditions, strong toxicity was observed in the citrinin heated at 140°C, this cytotoxicity becoming weaker by heating at higher or lower temperatures than 140°C and disappearing completely by heating at 170°C.

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We screened for immunoglobulin production stimulating factors (IPSFs) in polysaccharides using human-human hybridoma cells, HB4C5, cultured in serum-free medium. Among polysaccharides, citrus pectin, locust bean gum, and chitosan stimulated IgM production of HB4C5 cells. Especially chitosan showed the strongest IPSF activity ; 100 ng/ml of chitosan stimulated IgM production approximately 5-fold. Chitosan had several characteristics as IPSF, as follows. 1) For the IPSF activity, 70-90% deacetylation was essential. 2) Chitosan oligomers (n=5, 6, 7) and chitin oligomers (n=5, 6, 7) showed no IPSF activities. 3) The IPSF activity of chitosan was inhibited by glucosamine, one of the constitutive sugars of chitosan. 4) Chitosan stimulated IgM production of human lymphocytes in serum-free culture, but not IgG or IgA, nor in serum-supplemented culture.

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A genomic library of Streptomyces sp. KB210-8SY, prepared in the plasmid vector pACYC184, was screened to obtain the gene encoding sarcosine oxidase with probes based on the amino acid sequence of the protein. A plasmid pSOXS13, which was isolated from a clone identified by hybridization with the probes, contained a 8.4-kb insert of Streptomyces DNA. When the 2.0-kb MIuI/EcoRV DNA fragment of pSOXS13 was inserted into the Streptomyces vector pIJ680 and introduced into S. lividans, the transformants produced 100-fold more sarcosine oxidase intracellularly than KB210-8SY. The nucleotides of the 1.7-kb fragment containing the sarcosine oxidase gene were sequenced. An open reading frame encoded a mature sarcosine oxidase consisting of 388 amino acids, with a calculated molecular mass of 42, 107 daltons.

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We have investigated culture conditions for production of dihydrofolate reductase by Escherichia coli harboring a high expression plasmid, pTP64-1. Sorbitol addition and pH control were effective for the production of the enzyme in a jar fermentor. The enzyme was purified from a cell-free extract by column chromatographies on DEAE-Cellulofine and Superose Prep12 and showed a single band on SDS-polyacrylamide gel electrophoresis. The reduction of 200 mM dihydrofolate to 6(S)-tetrahydrofolate, an intermediate for l-leucovorin synthesis, was complete in 2 hr under anaerobic conditions, using 1.5 units/ml of the purified enzyme.

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Several kinds of modified chymotrypsin were prepared with water-soluble acylating reagents, and their characteristics after hydrolyzing with unmodified chymotrypsin in aqueous-N, N'-dimethylformamide (DMF) media were compared. It was found that chymotrypsin (Csin), of which a 20% amino group was modified with a benzyloxycarbonyl group (Z(20)Csin), had more favorable characteristics than unmodified chymotrypsin with regard to hydrolytic activity in an aqueous DMF media. We also investigated the Z(20)Csin-catalyzed peptide synthesis in two different solution systems. In the one-layer system containing water and DMF, Z(20)Csin catalyzed the peptide bond formation in a higher yield than that by unmodifide chymotrypsin and enabled a synthetic reaction in even an 80% (v/v) DMF media, in which the hydrolytic reaction could not be carried out. Z(20)Csin catalyzed the condensation between some N-acyl amino acids or peptide derivatives and amino acids in 90% ethylacetate, 90% hexane or 50% benzene. This latter method employs a two-layer system, and the modified enzyme may be able to reduce the number of synthetic steps when preparing acyl peptides.

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A ganglioside GM₃ analog containing a hydroxymethyl group in the place of the carboxyl group in the Neu5Ac unit was synthesized, in order to investigate the relationship between the structure of sialic acid and the functions of gangliosides.

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The hydrolyzate of partially N-acetylated chitosan by Bacillus sp. No. 7-M chitosanase was separated by gel filtration on Bio-Gel P-2. Sugar compositions and sequences of the oligosaccharides were identified by exo-splitting with β-GlcNase, fast atom bombardment mass spectroscopy, and proton NMR spectroscopy. In addition to chitooligosaccharides, (GlcN)₂, (GlcN)₃, and (GlcN)₄, hetero-chitooligosaccharides such as (GlcN)₂·GlcNAc·(GlcN)₂, GlcN·GlcNAc·(GlcN)₃, (GlcN)₂, ·GlcNAc·(GlcN)₃, and GlcN·GlcNAc·(GlcN)₄ were detected. These results indicate that Bacillus sp. No. 7-M chitosanase is absolutely specific toward the GlcN·GlcN bonds in partially N-acetylated chitosan and at least three GlcN residues were necessary to the hydrolysis of chitosan by chitosanase.

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Nutritive values and effects on the digestive functions of chronic feeding of an amino acid (AA) diet were compared with those of feeding a casein diet used three strains of male weanling rats under conditions of maximum growth. We demonstrated that the responsiveness to the casein and amino acid diet were different between rat strains. Growth and food intake in the AA group were clearly less than those in the casein group in Tokushima-Wistar rats, both parameters were the same in SLC-Wistar-St rats, and only growth was less in Sprague-Dawley rats fed the AA diet. The weights of stomach and small intestine, and the ileum activities of sucrase in all strain rats fed an AA diet were higher than those rats fed a casein diet, which indicates that larger and longer chyme remaining in the lumen, and slower absorption of nutrients in the AA groups.

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Chitinases I and II were purified from the culture supernatant of Aeromonas sp. 10S-24 by ammonium sulfate precipitation, SP-Sephadex C-50 chromatography, Sephacryl S-200 gel filtration, and chromatofocusing. Both enzymes were most active at pH 4.0 and the optimum temperature for I and II were 50°C and 60°C. Chitinase I was stable at pHs between 4 and 9 and at temperatures below 50°C and chitinase II was stable at pHs between 5 and 7 and at temperatures below 45°C. The molecular weights were estimated by SDS polyacrylamide gel electrophoresis to be 112, 000 and 115, 000 for I and II respectively, while gel filtration showed the molecular weight to be 114, 000 for both types of the enzyme. The pIs for I and II were 7.9 and 8.1, respectively. The activities of both enzymes were inhibited by Ag⁺ and iodoacetic acid.

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Amylases (I and II) extracellularly produced by an imperfect fungus, Fusidium sp. BX-1 in a medium containing glycerol as a carbon source, were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The electrophoretical mobilities of amylase I and II on native and SDS-polyacrylamide gels exactly coincided with each other. Their molecular weights were estimated to be about 52, 000. The sugar contents of amylase I and II were 3.4 and 4.7%, and the pIs were 8.70 and 8.55, respectively. The K_ms of the enzymes for soluble starch were 0.053 and 0.044%. The actions of the enzymes on soluble starch, short chain amyloses, and maltose were examined. Amylase I and II are identified as being to an α-amylase and a glucoamylase, respectively.

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In an attempt to delineate the binding site(s) of monellin to the receptor by means of a structure-taste relationship, we synthesized four monellin analogues, [Asn^A16]-, [Asn^A22]-, and [Gln^A25]-, [Asn^A26]- monellin, which were 7500, 750, 2500, and 5500 times as sweet as sucrose on a weight basis, respectively. Among them, [Asn^A22] monellin and [Gln^A25] monellin were less sweet than monellin, and were susceptible to the HPLC conditions used. It can be concluded that Asp¹⁶, Asp²², Glu²⁵, and Asp²⁶ residues of the A chain did not participate in binding with the receptor, since the sweet taste was not removed by replacing the amino acid residues with Asn or Gln. It can also be concluded that Asp²² and Glu²⁵ of the A chain may have participated in intramolecular binding, as was pointed out by Kim et al., since exchanging Asp²² and Glu²⁵ of the A chain with Asn and Gln significantly decreased the stability in solution.

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A droplet of an oil-in-water emulsion of methyl linoleate in a saccharide or protein solution that contained with a surfactant, a stabilizer, or both was dehydrated by drying equipment for a single droplet that resembled a spray drier. The lipid exposed on the surface of dehydated samples was extracted and measured by gas chromatography. Gum arabic or gelatin without additives resulted in little lipid being exposed ; they were good entrapping agents. Little lipid was exposed with a pullulan solution containing lecithin, sugar ester, carboxymethylcellulose, or sodium caseinate but much was exposed with a maltodextrin solution containing any of the surfactants tested. When both the surfactant lecithin and the stabilizer xanthan gum were added to the emulsion prepared in a maltodextrin solution, lipid was not detected. The results suggested that effective entrapping agents of liquid lipids cause much emulsification, stabilize the emulsion (that is, they cause the continuous phase to be very viscous), and create a dehydrated matrix of fine, dense network layers.

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Infantile rats were fed over a period of 5 weeks according to plan ingesting 20% casein and protein-free diets alternately every few days, and were examined for their growth and changes in metabolic function from the viewpoint of protein nutrition. Three groups of rats fed with the two diets alternating every one, two or three days were almost equal in growth to one another. However, their growth rates increased intermittently to such an extent as to be half that of the control fed ad libitum with the 20% casein diet. Two groups of rats given the 20% casein diet for one day and followed by two or three protein-free days showed much more growth-lag than the former groups did. The extent of body weight gain seemingly responded to the amount of protein intake as a whole throughout the experimental period. A change in metabolic function was observed for the hepatic RNA/DNA ratio in the animals receiving the protein-free diet for a few days prior to sacrifice. A similar indication was shown as to decreased levels of branched-chain and aromatic amino acids in the plasma, which didn't reach their normal level even after subsequent feeding with the 20% casein diet. Urinary nitrogen and urea excretions, which were markedly lowered by ingesting the protein-free diet for a few days, gradually returned to the normal levels within a few days after feeding instead with the 20% casein diet. Some metabolic functions were appreciably affected by unbalanced feeding that resulted in a variation in protein nutrition.

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Previously, a new type II methanotrophic bacterium, Methylocystis sp. M (strain M), was isolated in our laboratory [H. Uchiyama et al., Agric. Biol. Chem., 53, 2903-2907 (1989)]. In this paper, metabolites resulting from the degradation of trichloroethylene (TCE) by strain M were studied with gas chromatography-mass spectrometry. Ttichloroacetic acid, dichloroacetic acid, and a small amount of 2, 2, 2-trichloroethanol were detected in the water-soluble fraction of the reaction mixture. These results suggest that the conversion of TCE to trichloroacetaldehyde via a Cl-shift reaction, followed by the formation of trichloroacetic acid and 2, 2, 2-trichloroethanol, as well as a spontaneous breakdown of TCE oxide, with subsequent formation of dichloroacetic acid, etc., is involved in the TCE degradation pathway of the methanotrophic bacterium.

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A fucoidanase from the hepatopancreas of Patinopecten yessoensis was purified by ammonium sulfate precipitation, anion exchange chromatography, isoelectric focusing, and gel chromatography. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The fucoidanase was practically free from α-L-fucosidase and arylsulfatase activities. The molecular weight of the enzyme was estimated to be 85, 000 by gel filtration on TSKgel G3000SW and 84, 000 by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The enzyme hydrolyzed fucoidan to produce sulfated oligosaccharides as the reaction products.

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A model for use in the estimation of the stability of emulsions in cream layers is proposed. Stability was assessed by the parameter a_c, defined as the ratio of the kinetic energy of an emulsion particle to that of a molecule. Based on the DLVO theory and Stokes' law, the kinetic energy of a particle needed for it to cross over a potential barrier was calculated by consideration of the balance of forces working on the particle. The a_c value was a useful index for estimation of the stability of emulsions, and represented the empirically known effects of the properties of particles and the medium on stability.

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The radioactivity of ¹⁴C-labeled adenosine, inosine, and guanosine fed to excised tea shoots was incorporated into caffeine at a high rate (about 50% within 24hr), after the rapid labeling of free nucleotides, while the radioactivity of xanthosine was directly incorporated into 7-methylxanthosine, and then into 7-methylxanthine, theobromine, and caffeine. Cell-free extracts from tea leaves also phosphorylated four purine nucleosides into the respective nucleotides. The phosphorylation rate was adenosine»inosine≥guanosine»xanthosine. A small amount of enzyme activity involved in the reaction from guanosine to xanthosine was detected, but the reaction from inosine to xanthosine was not observed. Based on these results, it is considered that the pathway leading to the formation of xanthosine from adenine nucleotides in caffeine biosynthesis is via AMP→IMP→XMP→xanthosine in tea plants, while there is a possibility that guanine nucleotides are converted to xanthosine via guanosine.

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A cytokinin-binding protein (CBP) was purified from a crude extract of etiolated mung bean seedlings by a protocol involving affinity chromatography on benzyladenine-linked Sepharose 4B, ion exchange chromatography on DEAE-Sephadex A50, and gel filtration on Sphacryl S-400. The molecular weight was estimatd to be about 200, 000 by gel filtration. CBP appeared as two bands corresponding to molecular weights of about 45, 000 and 48, 000 on SDS-polyacrylamide gel electrophoresis. The dissociation constant for benzyladenine was 7.5 x 1^-7 M. ¹⁴C-Benzyladenine-binding to CBP was reversible and could be inhibited by the addition of kinetin or trans-zeatin. Adenine, AMP, and ADP had no inhibitory effect on the binding of¹⁴C-benzyladenine to CBP but the addition of ATP to the assay mixture enhanced the binding.

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