Bacterial cells respond to monoamine compounds, such as tyramine, dopamine, octopamine, or norepinephrine, and induce the syntheses of tyramine oxidase encoded by tynA and monoamine oxidase encoded by maoA. These monoamine compounds also derepress the synthesis of atsA-specified arylsulfatase that is repressed by sulfur compounds. These complex mechanisms of regulons regulated by monoamine and sulfur compounds has been analyzed by cloning and characterization of genes that are involved in the repression and derepression of the synthesis of arylsulfatase. The atsA gene forms an operon with the atsB gene, which encodes an activator of the expression of atsA. The negative regulator gene for arylsulfatase was found to code for dihydrofolate reductase (folA). The maoA gene forms an operon with the maoC gene, which has similarity to a dehydrogenase involved in the tyramine metabolism. The moaF gene encoding a 30-kDa protein, which is induced by tyramine; also forms an operon with the moaE gene. Finally, the moaR gene, which is induced by monoamine, was found to play a central role in the positive regulation of the expression of the monoamine regulon (moa) including the atsBA, maoCA, moaEF and tyn operons. The moaR expression is subject to autogenous regulation and to cAMP-CRP control. The MoaR protein has a helix-turn-helix motif in its C terminus. Thus, the MoaR protein probably regulates the operons by binding to the regulatory region of the moa regulon.

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A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: v_c=([3-PGA]-[PG])/2 and v_o= [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.

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Dihydroflavonol taxifolin and its glycoside, astilbin, from Engelhardtia chrysolepis were evaluated as antioxidants and radical scavengers. These dihydroflavonols inhibited superoxide anion production in the xanthine/xanthine oxidase system. Microsomal lipid peroxidation induced by NADPH-cytochrome P-450 reductase was also inhibited by these flavonoids. Mitochondrial lipid peroxidation was inhibited only by the aglycon. Taxifolin protected peroxy radical-damaged mitochondria with no effect on enzyme activity. Furthermore, taxifolin and astilbin protected red cells against oxidative hemolysis. These dihydroflavonols were found to be effective for protecting subcellular systems and red blood cells against oxidative stress in vitro.

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A possible obstacle in the development of hybrid strains of Acinetobacter calcoaceticus by the introduction of a metabolic pathway into the chromosome is genetic instability of the resulting recombinant strains. Therefore, the possibility that the pobA gene can be used as a chromosomal cloning site where the transposed genes can be maintained and expressed, was explored in this study. For this purpose, two model hybrid strains of A. calcoaceticus were created, in which a DNA fragment carrying catBCIJFD genes for catabolic degradation of catechol was inserted into pobA in opposite directions of each other, and their genetic stabilities were experimentally examined. Our data demonstrated that the stability of the genes neighboring the insertions depends on the orientations of the insertions. Also, the data further indicated that the functional metabolic pathways introduced into pobA can be expressed successfully as far as the insertion is engineered in an appropriate way. Concurrently, it was proposed that the pobA can be used as a chromosomal cloning site, and that introduction of an useful metabolic pathway into pobA may offer considerable promise to the construction of a hybrid strain with improved metabolic capabilities.

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A polysaccharide, in which the main part of the side chains were depleted, was prepared from the acidic polysaccharides of Fusarium sp. M7-1 by digestion with lyase of Cellulomonas sp. and mild acid treatment. This polysaccharide was degraded into several fragments, neutral oligosaccharides, neutral polysaccharide, and acidic polysaccharide, by an enzyme, endo-β-galactofuranosidase, produced by Bacillus sp. The main components of the oligosaccharides were isolated and identified as Galfβ1→6Gal, Glcα1→2Galfβ1→6Gal, Glcα1→2Galfβ1→6Galfβ1→6Gal, Glcα1→2Galfβ1→(6Glcα1→2)Galfβ1→6Gal and Glcα1→2Galfβ1→6(Galfβ1→2)Galβ1→6Galβ1→6Gal. The molecular mass of the neutral polysaccharide fragment was estimated to be about 6000 Da by gel filtration chromatography. The polysaccharide fragment consisted of an α1→6 linked mannan main chain to which various sugars, namely Glc, Man, and Rha were attached through α1→3 (or 2) linkages. The molecular mass of the acidic polysaccharide fragment was estimated to be about 6000 Da from the amounts of the reducing terminal galactose. The chemical structures of the oligosaccharides derived from the acidic polysaccharide fragment by mild acid hydrolysis were identified as reported structural units [Iwahara et al., J. Biochem., 112, 355-359 (1992)]. The structure of the mild-acid-resistant part of the acidic polysaccharide fragment was assumed to be a polyuronide to which various sugars such as Glc, Man, and GlcNAc are attached as the side chains. The linkage modes of each sugar are not clear.

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The fatty acid composition of the lipid of yellowfin tuna (Thunnus albacares) caught in two different localities, Philippine Sea (a tropical zone) and the Pacific coast area of Japan (a temperate zone) is described. The total lipids of various organs (dorsal ordinary muscle, ventral ordinary muscle, dark muscle, liver, heart, pyloric cecum, and orbital region) and of the stomach contents were extracted, and the fatty acid comosition was analyzed by gas-liquid chromatography (GLC). Docosahexaenoic acid (DHA; 22:6n-3) was the major unsaturated fatty acid in the lipid of all organs in the specimens examined from both localities, the mean DHA content accounting for more than 25% (mean±S.D. of 26.9±5.7%) of the total fatty acids. This value is markedly different from the fatty acid profile of other fish species, because, in general, the fatty acid composition of other species is variable and the DHA content is less than 20% of total fatty acids. Although the mean DHA content of the total fatty acids in the lipid of yellowfin tuna caught in the tropical and temperate zones was markedly higher than that in other fish species, there was a small difference between that in the northern samples (temperate waters, 30.5±6.1%) and the southern samples (tropical waters, 25.9±5.2% ). It is suggested that this difference may be due to environmental effects, e.g., the fatty acid composition of the lipids of prey organisms, because there is also a small difference between the mean DHA content of northern prey fish (22.7±6.1%) and that of southern prey fish (19.2±4.0%).

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Glucose-6-phosphate dehydrogenase in a yeast, Hansenula mrakii IFO 0895 is induced when the cells are cultured in a medium containing lipid hydroperoxide. The enzyme was purified from H. mrakii to the homogeneous state on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be approximately 52 kDa by SDS-PAGE and 130 kDa by Sephadex G-150 column chromatography, respectively. The enzyme was specific to glucose-6-phosphate and NADP⁺, and K_m values for glucose-6-phosphate and NADP⁺ were 293 μM and 24.1 μM, respectively. The enzyme activity was inhibited by diethylpyrocarbonate and 2, 4, 6-trinitrobenzene sulfonate, and by metal ions such as Zn²⁺, Cd²⁺, Cu²⁺, and Al³⁺. tert-Butyl hydroperoxide, a kind of lipid hydroperoxide, slightly (approximately 20%) increased the enzyme activity.

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E. coli cells harboring an expression plasmid with a brain-derived neurotrophic factor (BDNF) gene obtained from rat BDNF cDNA were sonicated and centrifuged to obtain a precipitate containing BDNF. Ten different proteins of BDNF were purified from the precipitate and the three disulfide linkages of six proteins were identified. Those disulfide structures were different from that of authentic BDNF and the biological activities of BDNF with mismatched disulfide linkages (EC₅₀ of 2 to 15ng/ml) were much lower than that of authentic BDNF (EC₅₀ of 30 pg/ml). The BDNF with mismatched disulfide linkages inhibited the biological activity of authentic BDNF.

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Starch degradation was not increased under dark-anaerobiosis compared with that under dark-aerobic or light-anaerobic conditions in a green alga, Chlamydomonas sp. MGA161, though was increased in a control, C. reinhardtii. Orthophosphate (Pi) stimulated starch degradation and increased ethanol and glycerol excretion while decreasing H₂ and acetic acid formation in strain MGA161. Phosphofructokinase was little increased in cells treated with Pi. Incubation with Pi inhibited hydrogenase activity in intact cells, and was accompanied by stimulation of starch degradation. Addition of H₂ to the fermentative gas phase resulted in uptake of the H₂ and the disappearance of H₂ evolution, which in turn raised the rate of starch degradation while increasing the excretion of ethanol and glycerol and decreasing the release of acetic acid. It is suggested that the starch degradation is increased not in a state that the ATP neogenesis through proton motive force does not function in dark-anaerobiosis but in a state that the reducing equivalence is increased by H₂ uptake and repression of the release.

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Insulin-like growth factor-I (IGF-I) exerts its effect through the IGF-I receptor. To investigate the effects of nutritional status on chicken IGF-I receptor gene expression, a solution hybridization/RNase protection assay for IGF-I receptor mRNA was developed. A cDNA clone corresponding to the carboxyl-terminal region of the IGF-I receptor was obtained by reverse transcription-PCR (RT-PCR). Sequence analysis of the clone showed that this region of the chicken IGF-I receptor is highly divergent from the human IGF-I receptor. IGF-I receptor mRNA was detected in all tissues examined from newly hatched chickens. The rank order of the IGF-I receptor mRNA levels was liver<thigh muscle<stomach<heart<lung<kidney<brain. In 1-week-old chickens, 5 days of starvation caused a 2.5- to 3-fold increase in the mRNA in muscle and kidney. Starvation of 4-week-old chickens for 5 days caused a 1.7 to 2.2-fold increase in IGF-I receptor mRNA levels in kidney, liver, and muscle. In contrast, IGF-I receptor mRNA levels in brain failed to change. The mRNA levels were reduced to the control level by refeeding of the starved chickens for 24 h. These data suggest a tissue- and development-specific response of chicken IGF-I receptor gene expression to nutritional status.

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We measured xylanase activities upon eight chromogenic substrates, o- or p-nitrophenyl-β-D-xylopyranoside (oNP-X or pNP-X) and o- or p-nitrophenyl-β-D-xylooligosaccharides (oNP-X_n or pNP-X_n, n=2-4), and studied for their uses as substrates in a kinetic study. The K_cat and K_m of Bacillus pumilus xylanase (EC 3.2.1.8) activities for pNP-X₂ were 0.24 s<-1> and 0.5 mM, respectively. The relative xylanase activities with the other substrates to that with pNP-X₂, pNP-X, pNP-X₃, pNP-X₄, oNP-X, oNP-X₂, oNP-X₃, and oNP-X₄ were <0.001, 9.4, 9.7, <0.001, 19, 190, and 200, respectively. HPLC analysis of the digestion products of oNP-X₂ or pNP-X₂ showed that the xylanase hydrolyzed each of the substrates only at the ether bond between nitrophenol and xylobiose. All ether bonds of pNP-X₃, oNP-X₃, pNP-X₄, or oNP-X₄ were hydrolyzed by the xylanase and further hydrolyses proceeded in the digestion products, e.g., oNP-X₂ and oNP-X₃ from oNP-X₄. Therefore, oNP-X₂ was screened as a useful substrate in a kinetic study of the xylanase. The K_m and K_cat of the xylanase for oNP-X₂ were 0.38 mM and 2.29 s^-1, respectively. Thermodynamic studies showed that the higher reaction rate obtained with oNP-X₂ than that with pNP-X₂ was due to a significant decrease in the activation energy change, despite a decrease in the activation entropy change.

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Possible disaccharide fragments of the major component of HS-142-1, a novel polysaccharide antagonist for functional atrial natriuretic peptide (ANP) receptors, O-(4-O-caproyl-β-D-glucopyranosyl)-(1→6)-4-O-caproyl-D-glucopyranose (1) and O-(3-O-caproyl-β-D-glucopyranosyl)-(1→6)-3-O-caproyl-D-glucopyranose (2), were respectively synthesized in a stereo- and regio-controlled manner. Deprotection of 2, 2'-di-O-caproyl derivative 35 gave a complex mixture due to undesired acyl migration. In contrast, 2, 4'-di-O-caproyl analog 39 was successfully deprotected to give O-(4-O-caproyl-β-D-glucopyranosyl)-(1→6)-2-O-caproyl-D-glucopyranose (40).

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The cAMP pathway and the Ras pathway are the two major pathways to sexual development in the fission yeast Schizosaccharomyces pombe. To understand the cAMP pathway or the related pathway, we analyzed mutants that display a phenotype similar to cyrl^-, that is, hyper-sporulation. Nine mutants termed sam (sporulation abnormal mutant), which are highly inclined to sexual development despite the presence of nitrogen sources, were partially characterised. Cyclic AMP was detected in all nine sam mutant cells, and over-expression of the adenylyl cyclase gene (cyrl) failed to suppress the hyper-sporulation phenotype of these sam mutants, suggesting that none of the sam mutants were likely to be allelic to cyrl. Epistatic tests of sam mutants showed that they were divided into two dominant and seven recessive mutants. Dominants were able to make spores in sam/sam⁺ heterodiploid cells upon abundant nutrients. Both two dominant mutants bypassed the inability to make spores in ras1 deficient diploid cells, suppressed the deficiency to execute sporulation in byr2 deficient diploid cells, but failed to suppress the byr1 deficiency. Two dominant mutations seem not to occur within the byr2 gene.

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The effects of green tea tannin on nephrectomized rats were examined. There were increases in blood urea nitrogen, serum creatinine, and urinary protein, and a decrease in creatinine clearance in the nephrectomized control rats, whereas better results for these parameters were obtained in rats given green tea tannin after nephrectomy, demonstrating a suppressed progression of the renal failure. When the renal parenchyma was partially resected, the remnant kidney showed a decrease in the activity of radical scavenger enzymes. Green tea tannin, however, was found to lighten the kidney under such oxidative stress. Mesangial proliferation and glomerular sclerotic lesions, which were conspicuous in the rats that were not given green tea tannin after nephrectomy, were also relieved.

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When defatted soy milk was ultracentrifuged, 34 kDa allergenic soybean protein Gly m Bd 30 K was more abundant in the precipitate than in the supernatant by an SDS-PAGE analysis. The addition of more than 10 mM of 2-mercaptoethanol (2-ME) to the soy milk resulted not only in further removal of the 34 kDa allergenic protein to the precipitate, but also in better recovery of conglycinin in the supernatant. After a two-dimensional SDS-PAGE analysis (the first dimension, minus 2-ME; the second, plus 2-ME) of the precipitates, superimposition between the CBB-stained gel and the electroblotted membrane stained with a monoclonal antibody specific to Gly m Bd 30 K indicated that part of Gly m Bd 30 K was preferentially bound to the α'- and α-subunits of conglycinin, and that part of them had formed the dimer through a disulfide bond.

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Long-distance PCR was applied to rapidly map the restriction sites of long inserts cloned on λEMBL3 phage vector. The restriction sites of 9 of 15 enzymes were completely assigned in a model experiment within 14 h, including 8 h for the PCR amplification. This method was found particularly useful for genomic DNA cloning when the partial sequence of the corresponding cDNA is known.

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The fatty acid composition of lipids in various organs and in the stomach contents of two species of frigate mackerel, Auxis rocheri and Auxis thazard, related to the tuna species was determined. Docosahexaenoic acid was the dominant unsaturated fatty acid accounting for 20% or more of the total fatty acids in all organs of the two frigate mackerel species (mean±S.D.: 22.6±6.0% for A. rocheri, 28.0±4.3% for A. thazard), while the fatty acids in lipids from their stomach contents were comparatively low (1.5-13.0% for A. rocheri, 15.4-16.5% for A: thazard). It is suggested that the high content of DHA in the lipids of tuna species is a general characteristic.

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Bovine κ-caseinoglycopeptide (residues 106-169, CGP) completely inhibited the PHA-induced proliferation of mouse splenocytes when CGP was added simultaneously with PHA. The inhibitory effect, however, was reduced to about one-half when CGP was added after 24h of cultivating the splenocytes with PHA or when anti-IL-1ra antibody was added simultaneously with PHA and CGP. On the other hand, CGP bound to mouse CD4⁺ T cells but not to CD8⁺ T cells. CGP suppressed IL-2 receptor expression of the PHA-stimulated mouse CD4⁺ T cells.

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A host-vector system was constructed in Bacillus megaterium strain NK84-0128; an oxetanocin A producer. The replication origin of an endogeneous plasmid, P-4, was used to construct a potential plasmid vector, pSM5, which had a chloramphenicol resistance gene as a selective marker. Plasmid transformation by a protoplast method was used in B. megaterium strain NK84-0128. The maximum transformation frequency attained with the pSM5 plasmid was 2.0×10⁴ cfu/μg DNA.

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Protoplast fusion of Lactobacillus plantarum ATCC10012 was examined with an electric field fusion method using its double mutants having drug resistance and auxotrophic marker genes. Under the conditions of pulse width of 150 μs and an electric field intensity of 6 kV/cm, the frequency of obtained strains carrying the phenotypes from both mutants was highest (6.2×10^-7). The frequency was at least one order higher than the reversion frequencies and that of conjugation. These results suggest the completion of protoplast fusion.

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We examined the effects of n-3 polyunsaturated fatty acid (PUFA), such as α-linolenic (α-LA), eicosapentaenoic (EPA), and docosahexaenoic acid (DHA) on immunoglobulin (Ig) production by spleen lymphocytes of Sprague-Dawley rats. n-3 polyunsaturated fatty acid (PUFA) strongly inhibited the production of IgA and IgM and that of IgG weakly at 100 μM. When the lymphocytes were treated with n-3 PUFA in the presence of other inhibitory biomaterials such as lectins, some PUFA attenuated their inhibitory effect on Ig production. In the presence of concanavalin A (ConA), all n-3 PUFA attenuated the inhibitory effect of ConA on the production of IgM or IgG but increased its inhibition of IgA synthesis. Thus, the interaction of n-3 polyunsaturated fatty acid and lectins in spleen interfere with each other or the expression of Ig production regulating activity.

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Six chlorophyll-related compounds isolated from leaves of Neptunia oleracea (Leguminosae) inhibited the activation of tumor promoter-induced Epstein-Barr virus (EBV). Photo-irradiation of pheophorbide a, the major active constituent, did not enhance this inhibitory activity, and thus ruled out any photosensitizing effect playing the major role.

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Previously isolated cDNA clone A3-12 that was expressed in E. coli as the fusion protein with Trp E showed immunoreactivity with the mouse antibody raised against isolated α-globulin from rice seed. The N-terminal amino acid sequences determined for the purified α-globulin and its tryptic peptides were identical with the deduced amino acid sequence reported, except for two residues at the protein N terminus. An error in the reported sequence was confirmed by re-sequencing the cDNA, the nucleotide sequence for the two N-terminal residues being shown to be CA(GC)^^^TG and not CA(CG)^^^TG. Thus, the protein encoded by cDNA clone A3-12 was identified to be the major rice seed globulin, α-globulin, with an apparent molecular mass of 26 kDa.

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Sweet potato β-amylase catalyzes two different kinds of reactions:hydrolysis of amylose, etc. and hydration of maltal (α-D-glucopyranosyl-(1→4)-2-deoxy-D-glucal). To investigate whether both the reactions are catalyzed at the same catalytic site or not, an inhibition study on both the reactions and an affinity-labeling of Glu187 were done using 4-O-α-D-glucopyranosyl-(1→4)-1-deoxy-nojirimycin (GDN) as an inhibitor. As GDN showed competitive inhibition with the same K_i for these reactions, it was concluded that both the reactions occur at the same catalytic site.

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The polyether-macrolide antibiotic, boromycin, was isolated as a potent anti-human immunodeficiency virus (HIV) antibiotic from a fermentation broth of Streptomyces sp. A-3376. Boromycin was found to strongly inhibit the replication of the clinically isolated HIV-1 strain as well as the cultured strain in in vitro laboratory experiments. The mechanism for the anti-HIV activity of boromycin is suggested to involve blocking the later stage of HIV infection, and probably the maturity step for replication of the HIV molecule.

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Enterococus faecalis plasmid pAM373 encodes a mating response to the sex pheromone, cAM373, which is secreted from pAM373-free E. faecalis. cAM373-like activity was detected in a culture filtrate of Staphylococcus aureus. The major active substance, termed staph-cAM373, was isolated, and its structure was identified as a H-Ala-Ile-Phe-Ile-Leu-Ala-Ala-OH heptapeptide.

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The effect of fourteen quaternary ammonium iodides on ent-kaurene synthase from Cucurbita maxima L. was examined. Structure-dependent inhibition of the overall AB activity of ent-kaurene synthase was found, but only weak or no inhibition on B activity, suggesting that the site of inhibition was ent-kaurene synthase A. N, N, N-Trimethyl-1-methyl-(2', 2', 4', 6'-tetramethyl-cyclohex-3'-en-1'-yl)prop-2-enylammonium iodide, which has structural similarity to geranylgeranyl pyrophosphate, reduced AB activity to 50% of the control value at a concentration of 0.1 μM.

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The antioxidant behaviors of vitamin E and its analogues, 2, 2, 5, 7, 8-pentamethyl-6-hydroxychroman and 1, 2-diacyl-sn-glycero-3-phospho-2'-(hydroxyethyl)-2', 5', 7', 8'-tetramethyl-6'-hydroxychroman, were studied in unilamellar vesicles. The two analogues scavenged aqueous radicals generated from azo compounds more efficiently than vitamin E. On the other hand, vitamin E scavenged the lipid peroxyl radicals preferentially. It is concluded that the superior antioxidant activity of vitamin E is attributed to its location suitable for breaking the chain propagation reaction.

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Phytoalexins are antimicrobial secondary metabolites which accumulate in plants against fungal invasion. Their production is triggered not only by fungal invasion, but also by a variety of elicitors. In rice plants, we have shown that CuCl₂ is a potent abiotic elicitor. Jasmonic acid has recently become known to play an important role in secondary metabolite production in plants at the cellular level. This led us to speculate, in CuCl₂-elicited rice leaves, that JA might also play an important role as a signal transducer for phytoalexin production. The endogenous level of JA increased rapidly in CuCl₂-elicited rice leaves, and exogenously applied JA caused a large amount of phytoalexin production in rice leaves. This phytoalexin production by CuCl₂ decreased when rice leaves were treated with JA biosynthesis inhibitors, but that by JA did not. JA is thus suggested to play an important role in the elicitation process leading to phytoalexin production in rice leaves.

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Bioassay-guided fractionation of the MeOH extract of Ornithogalum saundersiae bulbs led to the isolation of a new cholestane bisdesmoside with potent cytotoxic activities toward leukemia HL-60 and MOLT-4 cells. The structure was deduced mainly from spectroscopic information.

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