There is now growing interest in the functional role of adenosine A_2A receptors. Their distribution within the brain is restricted in the basal ganglia, particularly abundant in the striatum, which are thought to play a crucial role in the control of motor behavior. Indeed, newly developed A_2A receptor selective antagonists have a profound influence on motor functions, with anti-Parkinsonian activities in several animal models. Striatal spiny neurons serve as a major anatomical locus for the relay of cortical information flow through the basal ganglia. The GABA releasing projection neurons represent the A_2A receptor-mediated main target of adenosine. The GABAergic synaptic neurotransmission is regulated by adenosine via A_2A receptors on the presynaptic terminals. Blockade of this modulatory function by A_2A antagonists could repair striatopallidal abnormal neuronal activities provoked by striatal dopamine depletion in the Parkinsonian state. A_2A receptor antagonists provide a novel therapeutic potential for the treatment of Parkinson’s disease.

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Novel herbicidally active sulfonamide compounds having a 2-arylsubstituted oxiranylmethyl structure are racemates due to a chiral carbon in the oxirane moiety. To clarify the stereochemical structure-activity relationship, we synthesized each enantiomer of 4-chloro-N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-3,N-dimethylbenzenesulfonamide and N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-N-methyl-5,6,7,8-tetrahydronaphthalene-2-sulfonamide by chemical methods including Sharpless asymmetric chlorohydroxylation. The results of herbicidal tests indicated that the (S)-isomers were the active forms.

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Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken β2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.

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(+)-Magnostellin C, which is a tetrahydrofuran type of lignan bearing a chiral secondary benzylic hydroxy group, was stereoselectively synthesized from L-arabinose by using threo selective aldol condensation.

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As a model synthesis on cis-2,3-dibenzyl-4-butanolide lignan, cis-(2S,3R)-parabenzlactone bearing a chiral benzyl alcohol moiety was stereoselectively synthesized from L-arabinose.

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A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC). A fungus, Penicillium citrium, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia. ACC synthase, responsible for ACC synthesis in P. citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA. The ACC synthase from P. citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme. The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P. citrinum. Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation. The sequence of ACC synthase from P. citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.

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A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35°C in the presence of 5 mM Mn²⁺ and 0.5% (w/v) Triton X-100. Acid-soluble low-M_r (average M_r 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (M_r 70,000). The apparent Michaelis constants for UDP-GalA and low-M_r PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-¹⁴C-GalA and the low-M_r PGA yielded ¹⁴C-GalA₂ as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous α-1,4-linkages.

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In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5∼5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.

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The gene encoding the endo-β-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequneced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, sitedirected mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.

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The α-glucosidase gene (agdA) of Aspergillus nidulans has a single CGGN₈CGG type AmyR binding site in its promoter region. The binding site is functional in vivo as a cis-element responsible for induction by starch, and mutational studies indicated that both the CGG triplets are required for high-level induction. A part of AmyR (residues 1-411; AmyR_1-411), which was produced as a MalE fusion protein in E. coli, bound to the CGGN₈CGG site of the agdA promoter. DNA binding profiles to the mutant binding sites that lacked both or either one of the CGG triplets suggested that AmyR_1-411 can bind to a single CGG triplet site with low affinity and that two AmyR molecules cooperatively bind to the CGG direct repeat.

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An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by post-translational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.

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The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.

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Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (Δsyr1/erg3 null mutation). The sensitivity of the indicator strain (Δsyr1/erg3Δpdr5Δsnq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (∼0.5 μg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.

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Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.

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α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high K_m for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k₀ values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K_m values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (A_i’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A_i value at subsite 3 was larger than that of subsite 1.

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Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB. The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.

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Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.

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Anti-viral activities of Agaricus blazei Murill were investigated. The water extracts of the cultured mycelia and fruiting bodies were fractionated with different concentrations of ethanol. To several viruses which have cytopathic effects (CPE) on VERO cells, inhibition of these effects by the ethanol fractions was tested. Strong inhibition of CPE induced by western equine encephalitis (WEE) virus was observed in the mycelial fractions but not those of fruiting bodies.

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We isolated a cDNA for basic class I chitinase (ChitiWb1). ChitiWb1 cDNA encodes a protein that consists of 315 amino acid residues and has a signal peptide. Northern blot analysis indicated that the class I chitinase mRNA in leaves and cultured cells of winged bean was increased by treatments with NaCl, KCl, CaCl₂, mannitol or saccharose, but not with abscisic acid. Thus, class I chitinase expression was shown to be up-regulated by osmotic stress.

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An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (M_r=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C₃-C₄) esters are the most preferred substrates. The enzyme is inhibited by HgCl₂ and p-chloromercuribenzoate but not by phenylmethyl-sulfonyl fluoride.

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Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.

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Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This “phyto-fermentation” system can be used in plant production of various chemical commodities and pharmaceuticals.

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Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HνNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.

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α-D-Glucosylglycerol (GG) is a mixture of 2-O-α-D-glucosylglycerol (GG-II), (2R)-1-O-α-D-glucosyl-glycerol (R-GG-I) and (2S)-1-O-α-D-glucosylglycerol (S-GG-I). GG has been found to be slightly hydrolyzed in vitro only by rat intestinal enzymes, but hardly at all by other digestive juices. GG suppressed the hydrolysis of maltose, sucrose and isomaltose by rat intestinal enzymes because the amount of glucose in the digestion of a mixture of GG and disaccharide was less than the sum of that in each individual digestion. The consumption of GG was suppressed by isomaltose, but promoted by maltose, with the hydrolysis of GG being suppressed. Sucrose appeared to suppress only the consumption of S-GG-I, suggesting that S-GG-I was hydrolyzed by the active site of sucrase in a sucrase-isomaltase complex. Transglucosylation seems to have occurred more frequently in the individual digestion of maltose and isomaltose than in that of GG and sucrose. GG seemed to promote transglucosylation in the presence of maltose, to suppress it with sucrose, and to delay it with isomaltose.

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The effects of dietary oils on stress-induced changes in the liver glycogen metabolism of male Wistar rats at 6 weeks of age were investigated. The rats were subjected to repetitive water-immersion restraint and fed with a 20% saturated fatty acid mixture (PSC), olive oil (OLI), safflower oil (SAF), or linseed oil (LIS) diet. Stress loading decresed the body weight gain, although the food intake was hardly changed, and the weights of the liver and spleen generally declined regardless of the elapsed time after stress loading and the type of dietary oil. The adrenal weight was generally enhanced by stress in all deitary groups, and particularly tended to be greater in the OLI and PSC groups than in the other two. The plasma corticosterone concentration increased immediately after stressing (Stress-1), but approached the level of the rats with no stress (No stress) 2 h after releasing the stress load (Stress-2) in all groups. The enhancement of corticosterone level in the Stress-1 animals was large in the PSC and OLI groups, and the decline of this level in the Stress-2 animals was small in the OLI group when compared with the other groups. Although the concentrations of total cholesterol (T-CHOL) and triacylglycerol (TG) in the plasma were decreased by stress loading in all groups, these concentrations in the PSC and OLI groups were nearly always higher than in the other groups. The liver serine dehydratase (SDH) activity enhanced by stress was high in the OLI group and tended to be high in the PSC group when compared with the other groups. The contents of liver glycogen were reduced in the Stress-1 animals and extremely elevated in the Stress-2 animals of all groups, and particularly in the OLI group, the reduction in the Stress-1 animals was smaller and the enhancement in the Stress-2 animals was greater than in the other groups. These results suggest that feeding oleic acid to rats exposed to water-immersion restraint further accelerated liver glycogen synthesis through the rise in liver SDH activity due to inceased corticosterone secretion when compared with the effect from linoleic and α-linolenic acids.

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Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94%. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.

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The antimutagenicity of the 3-sophoroside-5-glucoside of cyanidin and 3-sophoroside-5-glucoside of peonidin, the anthocyanin derivatives deacylated from the 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and 3-(6,6'-caffeylferulyl-sophoroside)-5-glucoside of peonidin (YGM-6) which had been purified from the sweetpotato with purple-colored flesh, was investigated by using Salmonella typhimurium TA 98. A comparison of the antimutagenicity between YGM-3 and YGM-6 and the deacylated derivatives showed that the activity of cyanidin was stronger than that of peonidin. Deacylation of the peonidin-type pigment markedly decreased this antimutagenicity. Caffeic acid showed the strongest antimutagenicity of the constituent organic acids of the anthocyanin pigments, caffeic acid, ferulic acid, and p-hydroxybenzoic acid. These results suggest that the cathecol structure plays an important role in the strong antimutagenicity of anthocyanin pigments.

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A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R^*,4R^*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R^*,4R^*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC₅₀ of the compounds were 4.0×10^-6, 4.9×10^-6, 9.4×10^-6, and 5.1×10^-6M, respectively.

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The anti-inflammatory and anti-allergic activity of perilla leaf extract was investigated. The oral administration of perilla leaf extract to mice inhibited two types of acute inflammatory models, arachidonic acid-induced ear edema and 12-o-tetradecanoylphorbol-13-acetate-induced ear edema. Oral administration of perilla leaf extract also inhibited the contact dermatitis model, oxazolone-induced ear edema, by affecting sensitization.

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Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.

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A strict anaerobic bacterium, strain Y51, was isolated from soil contaminated with tetrachloroethene (PCE). Strain Y51 is capable of very efficiently dehalogenating PCE via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at concentrations as high as 960 μM and as low as 0.6 μM. Strain Y51 was Gram-negative, motile with some lateral flagella, and curved rodshaped. On the basis of the 16S rDNA sequence, the organism was identified to be a species within the genus Desulfitobacterium. Strain Y51 also had dehalogenation activities toward polychloroethanes such as hexa-, penta-, and tetrachloroethanes, from which dichloroethenes were produced as the final products. The cell extracts mediated the dehalogenation of PCE with reduced methyl viologen as an electron carrier at the specific rate of 5.0 nmol·min^-1·mg cell protein^-1 (pH 7.2, 37°C). Dehalogenation was highly susceptible to air oxidation, and to potential alternative electron acceptors such as nitrite or sulfite.

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A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.

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Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidulans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS(fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.

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Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.

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Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis

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A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of th 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.

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Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of V_{max(oxaloacetate)}/V_max(malate) and K_cats (555 s^-1 for oxaloacetate, 481 s^-1 for NADH) of MDH-M2 were higher than the ratio (1.37) of V_max and K_cats(241 s^-1 for oxaloacetate, 271 s^-1 for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD⁺ and the activity of MDH-M1 by oxaloacetate.

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The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).

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