Two isozymes of γ-glutamyltranspeptidase, GGT-A and GGT-B, were purified to elettrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(γ-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23, 000 (I), 39, 000 (II), and 40, 000 (III). GGT-B was composed of two subunits with molecular weights of 22, 000 (I) and 39, 000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55°C) but showed a signiticantly different thermal stability at 55°C. Both GGT-A and -B used D-γ-glutamyl-p-nitroanilide as well as the L-isomer as the γ-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.

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Methods for estimating the parameters of nonlinear adsorption isotherms of Langmuir and Freundlich types from a pulse response curve are proposed here based on the migration rate of an adsorbate at a constant concentration and the mean residence time of the adsorbate in a bed. The methods were used to estimate the parameters in isotherms for various combinations of adsorbent and adsorbate. The isotherms estimated by the proposed methods were compared with those estimated by conventional methods. It was demonstrated that the proposed methods could evaluate the parameters with fairly good precision when the type of isotherm was known. The criteria for discriminating the type of isotherm from the pulse response curve are also described.

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The antioxidative effect of quercetin was exrimined in metmyoglobin-induced oxidation of a fish oil-bile salt emulsion (average diameter of particles; 2.0μm) to evaluate its effectiveness during the digestion of highly oxidizabile oils. The activity of quercetin increased with the lowering of the initial peroxide value (PV) of the oil and its effectiveness was superior to that of α-tocopherol. A synergistic antioxidant effect was observed upon the addition of quercetin and α-tocopherol irrespective of the initial PV of the oils, and quercetin was consumed faster than α-tocopherol. The loss of quercetin was larger than that of α-tocopherol when cumene hydropeioxide and metmyoglobin were mixed in a trimyristin-bile salt emulsion. In an ultrafiltration experiment on emulsitied oil with a membrane filter of 100 nm pore size, the recovery of quercetin in the filtrate was higher than that of α-tocopherol. These data suggest that quercetin was an antioxidant in the digestion of fish oil. The effectiveness seems to come from its distribution in the emulsitied oil, diffrient ffom that of α-tocopherol, and its ability to scavenge radicals generated from the reaction of lipid hydfoperoxides with metmyoglobin.

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The interaction of inorganic mercury with human and bovine milk proteins was studied. Gel filtration chromatography of skimmed milk and whey incubated with mercury showed that, in human milk, mercury was mainly bound to caseins, while a low proportion was bound to albumin. In bovine milk, mercury was associated with two protein fractions, caseins and β-lactoglobulin. Furthermore, it was shown by electrophoresis that mercury induced the formation of dimers of β-lactoglobulin. Thus, in both human and bovine milk, mercury prossessed greater ability to interact with milk proteins than to the low-molecular-weight substances. However, the pattern of mercury distribution was different between the milk of these two species.

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The antioxidant and free radical scavenging effects of dopamine, noradrenaline, tyramine, and tyrosine were investigated and compared with α-tocopherol. The antioxidant effect of dopamine and its related compounds on peroxidation of linoleic acid were in the order of dopamine > α-tocopherol = tyramine > tyrosine > noradrenaline as measured by the thiocyanate method. These amine compounds had reducing power, and a scavenging effect on reactive oxygen species, i.e., superoxide anion and hydroxyl radical. The results for reducing power and scavenging effect of these amine compounds had a similar trend as their inhibition of linoleic acid peroxidation. The antioxidant activity of these amine compounds in soybean oil was also evaluated by the Rancimat method. The induction time to reach 100 meq/kg peroxide value (POV) of soybean oil for dopamine, α-tocopherol, tyramine, tyrosine, noradrenaline, and control were 9.0, 8.2, 8.0, 6.4, 4.6, and 4.3 h, respectively. The antioxidant efficacy of amine compounds seems to be correlated with the numbers of hydroxy groups and their position on the phenolic ring.

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Novel 25-substituted milbemycin A₄ derivatives were synthesized from 25a-hydroxymilbemycin A₄ and 25b-hydroxymilbemycin A₄, which had been obtained by the microbial oxidation of milbemycin A₄. The acaricidal activity of each synthesized derivative was tested against Tetranychus urticae, and all of the synthesized derivatives showed higher activity than parent milbemycin A₄. Some of the derivatives had higher acaricidal activity than milbemycin D, which had higher acaricidal activity than milbemycin A₄. Among them, 25b-methylmilbemycin A₄ was the most active derivative, with 100% mortality of the mite at a concentration of 1 ppm, and 63% mortality at 0.1 ppm.

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Chattonella marina, a raphidophycean flagellate, is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide (O^-₂), hydrogen peroxide (H₂O₂), and hydroxyl radicals (·OH), which may be responsible for the toxicity of C. marina. In this study, we found that other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa jqponica also produce O^-₂ and H₂O₂ under normal growth condition. Among the flagellate species tested, Chattonella has the highest rates of production of O^-₂ and H₂O₂ as compared on the basis of cell number. This seems to be partly due to differences in their cell sizes, since Chattonella is larger than other flagellate species. The generation of O^-₂ by these flagellate species was also confirmed by a cemiluminescence assay by using 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo [1, 2-a] pyrazin-3-one (MCLA). All these raphidophycean flagellates inhibited the proliferation of a marine bacterium, Vibrio alginolyticus, in a flagellates/bacteria co-culture system, and their toxic effects were suppressed by the addition of superoxide dismutase (SOD) or catalase. Our results suggest that the generation of reactive oxygen species is a common feature of raphidophycean flagellates.

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The fractal structure of the aggregates in food protein gels was analyzed. Three kinds of food protein gels were prepared: (1) β-lactoglobulin (β-LG) gel; (2) 11S soybean globulin gel; and (3) caseinate gel. From the concentration dependence of the gel elasticity, the fractal dimensions D_f of the aggregates in the gels were evaluated, according to the theory of Shih et al. These gels showed the weak-link behavior described in the theory of Shih et al. The values obtained for D_f were 2.6-2.7, which were larger than those predicted by the cluster-cluster aggregation model for a dilute system. In addition, for the β-LG gels, the fractal dimension was also evaluated from the analysis of the gel image obtained with a confocal scanning laser microscopy, the value being close to that evaluated from the concentration dependence of the gel elasticity. These results indicate that the elastic behavior of the aggregate gels is a reflection of fractal structure of the aggregates in the gels.

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Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence hris been developed by producing reDer fI E(-1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(-1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(-1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(-1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.

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Shikonin production by Lithospermum cell cultures is induced by transferring the cells into production medium. Six phenolic compounds, p-hydroxybenzoic acid, caffeic acid, sinapic acid, ferulic acid, syringaldehyde, and salicyclic acid, were detected in both shikonin-producing and non-producing cells. Their contents in the former were much lower than those in the latter except for salicylic acid, the content of which strongly increased when cells were producing shikonin. The cell wall fraction, after alkaline hydrolysis, gave two phenolic compounds, p-hydroxybenzoic acid and caffeic acid. Their contents were much higher in shikonin-producing cells than in shikonin-free cells. Of these compounds, exogenous addition of p-hydroxybenzoic acid increased shikonin production in the production medium. Although it is a precursor of shikoning the increment of shikonin produced was much larger than the administered p-hydroxybenzoic acid, suggesting this compound has a stimulatory effect on shikonin biosynthesis at a low concentration.

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We investigated the effects of feeding with a diet containing pyrazinamide (PYR) on the metabolism of L-tryptophan (Trp) to nicotinamide in streptozotocin (STZ)-diabetic rats and whether the diabetic action of STZ is prevented by feeding with the PYR diet, which is known as an inhibitor of aminocarboxymuconate-semialdehyde decarboxylase and poly(ADP)ribose synthetase and therefore, signiticantly increases the formation of nicotinamide from Trp in normal rats. As was expected, feeding with the PYR diet to the STZ-injected rats caused a signiticantly increased excretion of nicotinamide and its metabolites like that in normal rats. The body weight increased in the STZ-injected rats fed with the PYR diet, while it was lost in the STZ-injected rats fed with the non-PYR diet. However, the blood glucose level and the urinary excretion of glucose were not improved even when the rats were fed with the PYR diet. Therefore, it was suggested that chronically increasing the formation of nicotinamide from Trp could not completely prevent the STZ-diabetic action.

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The large scale fermentation of γ-polyglutamic acid (γ-PGA) by Bacillus subtilis (natto) was done using a 30-liter jar fermenter. A stable cultivation without foaming could be done with addition of 3% NaCl to the medium. The γ-PGA productivity became higher with increasing speed of agitation and amounts of glutamic acid added to the broth. Finally, we were able to obtain about 35 mg/ml of γ-PGA under the optimum conditions. The glutamic acid added to the medium was efficiently converted into γ-PGA in the stationary phase. To discover the role of L-glutamic acid added to the medium for γ-PGA biosynthesis by Bacillus subtilis (natto), the radioactivity incorporated into γ-PGA from ¹⁴C-L-glutamic acid was measured. As a result, radioactive γ-PGA was detected in the medium. Then, the glutamic acid in the medium was transported into the cells and actually polymerized as the glutamic acid unit of γ-PGA.

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Thermostable dipeptidase from Bacillus stearothermophilus, a typical metalloenzyme containing 1.0 g atom of Zn per mole of subunit of the dimeric enzyme was markedly activated by exogenous divalent metal ions such as Mn²⁺, Co²⁺, and Cd²⁺. In contrast, several others including Ba²⁺, Hg²⁺, and Cu²⁺ considerably, inhibited the enzyme, even the inherent metal, Zn²⁺, being slightly inhibitory. To study the metal-binding properties of this dipeptidase, the enzyme was completely resolved to the inactive, Zn-free apoenzyme by treatment with EDTA in the presence of guanidine hydrochloride in a weakly acidic buffer. The apoenzyme was readily reconstituted by incubation with either Zn²⁺, Mn²⁺, or Co²⁺, restoring the catalytic activity. The Mn-reconstituted enzyme had nearly twice the activity of the original Zn-enzyme. Combined with kinetic analyses of reconstitution of the apoenzyme with metal ions, these results show that the enzyme has two non-identical metal-binding sites, each with a different property. Furthermore, substitution of Mn²⁺ or Co²⁺ for Zn²⁺ considerably lowered the thermostability of the enzyme without affecting the overall conformation of the enzyme protein, suggesting that the prosthetic Zn is playing dual roles in conformational stability and catalysis of the thermostable dipeptidase.

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The oxidation of L-ascorbic acid (ASA) by molecular oxygen was studied in the absence of heavy metal ion catalysts. The formation of superoxide anion was confirmed during the autoxidation of ASA not only in aqueous solution but also in MeOH. The formation mechanism of superoxide anion was discussed based on both experimental and molecular orbital (MO) calculation results. It was proposed that ASA autoxidation proceeded via the C(2) okygen adduct of ASA, and superoxide anion would be directly released from the C(2) oxygen adduct of ASA, forming monodehydro-ASA (MDASA).

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(R, Z)-7, 15-Hexadecadien-4-olide, the sex pheromone of the yellowish elongate chafer (Heptophylla picea), was synthesized from L-malic acid in 15 steps. The synthetic pheromone was identical with the natural product in its MS, IR, GLC retention time, and biological activity.

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A sorbitol oxidase (SOX) was found in the cell-free extract of a strain isolated from soil. The strain was classified and designated as Streptomyces sp. H-7775. SOX is constitutively expressed in the cell. The molecular weight of SOX that purified from the cell-free extract was 45, 000. The optimum pH and the K_m for sorbitol were 6.5-7.5 and 0.26 mM, respectively. The prosthetic group was a covalently bound FAD. SOX catalyzed oxidation of D-sorbitol to glucose and hydrogen peroxide without any requirements of exogenous cofactors. SOX did not react with glucose, a reaction product of D-sorbitol. This feature is useful in its application for diagnosis.

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A bacterial strain, GS-9, isolated from soil as a levan-degrading microorganism produced an extracellular enzyme that converted levan into DFA IV. This strain was identified as Arthrobacter nicotinovorans. The DFA IV-producing enzyme was specifically induced by levan. The enzyme was purified 60-fold from culture supernatant to give a single band on SDS-PAGE. The molecular weight of this enzyme was 52, 000 by SDS-PAGE and a monomer by gel filtration. The enzyme gave DFA IV as a main product (>75%), and fructose, levanbiose, and two unidentified oligosaccharides as minor products, and was identified as a novel levan fructotransferase.

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To obtain genes with sequence similarity to aminopeptidase T (AP-T) of Thermus aquaticus YT-1, we cloned the genes encoding aminopeptidase Th (AP-Th) from Thermus thermophilus HB8 and aminopeptidase II (APII) from Bacillus stearothermophilus NCIB8924. The AP-Th gene encoded a polypeptide of 408 amino acid residues and the deduced molecular weight of this subunit was 45, 015. The APII gene encoded a polypeptide of 413 amino acid residues with a deduced molecular weight of 46, 207. The extent of amino acid sequence similarity between AP-Th and AP-T was 86%, and that between APII and AP-T was 43%. The substrate specificities of these expressed enzymes were similar, and each efficiently hydrolyzed leucyl- or phenyl-peptide substrates. Since the deduced amino acid sequence of these enzymes show no similarity to other known aminopeptidases, they appear to comprise an independent family of peptidases, designated the AP-T family. However, a conserved region within the enzymes of the AP-T family shows similarity to the active site signature of the leucyl aminopeptidase family, suggesting that these enzymes may belong to the leucyl aminopeptidase superfamily.

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Increase in endurance swimming capacity caused by capsaicin (CAP), a pungent component of red pepper, -induced increase of fat metabolism in mice was investigated using an adjustable-current water pool. The mice administered CAP via a stomach tube, showed longer swimming time until exhaustion than the control group of mice, in a dose-dependent manner. The maximal effect was observed at a dose of 10 mg/kg while more than 15mg/kg had no effect. The increase of endurance was observed only when CAP was administered two hours before swimming. After the administration of CAP, the serum glucose concentration rapidly increased and then decreased within 60 min, while the concentration of serum-free fatty acids gradually increased through 3 hours. The residual glycogen concentration of the gastrocnemius muscle after 30 min of swimming was significantly higher in the CAP-administered mice than in control mice, suggesting that use of the serum free fatty acids spared muscle glycogen consumption. The serum adrenaline concentration significantly increased with twin peaks at 30 min and two hours after administration of CAP. An experiment using adrenalectomized mice was done to confirm that the effect of CAP is due to increased energy metabolism through the secretion of adrenaline from the adrenal gland. The swimming endurance capacity of the adrenalectomized mice was not increased by CAP administration, although adrenaline injection induced a 58% increase in the endurance time. These results suggest that the increase of swimming endurance induced by CAP in mice is caused by an increase in fatty acid utilization due to CAP-induced adrenal catecholamine secretion.

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The OPC homologous series was synthesized from 2-[(Z)-2-pentenyl]cyclopenten-1-one in short steps and with high yields. The carbon-carbon bond formation was achieved by the 1, 4-conjugate addition approach. This method makes it possible to supply a sufficient amount of OPC homologues which would enable significant information to be collected for plant physiological studies.

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The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg²⁺. β, γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, And PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg²⁺, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg²⁺ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3'-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

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Two unidentified sugars were isolated from rose petals using HPLC. The isolated compounds were identified as methyl β-glucopyranoside and xylose using ¹H-NMR, ¹³C-NMR, and GC-MS. Methyl β-glucopyranoside and xylose were distributed in three cultivars tested relatively in large amounts. These results indicate that methyl β-glucopyranoside and xylose occur universally as soluble sugar constituents in roses.

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The oxidative stability of phosphatidylcholines (PCs) from soybean, chicken egg, and salmon egg in liposomes was compared with that in aqueous micelles. When each PC was oxidized in aqueous micelles, salmon egg PC was the most oxidatively stable, followed by chicken egg PC and soybean PC, however, no significant difference in the oxidative stability was apparent between chicken egg PC and salmon egg PC in liposomes. The main molecular species of soybean PC was 1, 2-dilinoleoyl-PC, while most of the PUFAs in chicken egg PC and salmon egg PC were not esterified at the sn-1 position but at the sn-2 position. Therefore, it is suggested that the oxidative stability of PC liposomes would be strongly influenced by the positional distribution of PUFAs in the PC molecule. Further studies on the oxidation of PC liposomes showed that chicken egg albumin and soybean protein protected PC bilayers against attack by free radicals generated in the aqueous phase.

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Konjac glucomannan degrading enzyme was purified to homogeneity from the culture broth of an anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group. The enzyme was composed of a single polypeptide chain with a molecular weight of 50, 000-53, 000. The enzyme was an endo-β-mannanase that acted specifically on the polysaccharides such as konjac glucomannan and coffee mannan, producing exclusively their smaller oligosaccharides and the monosaccharides. The optimal pH of the enzyme for the hydrolysis of konjac glucomannan was around 7-8 and the enzyme was stable in rather alkaline pH range of 8-10. The enzyme reaction was activated by the addition of CaCl₂ and dithiothreitol. It was suggested that the enzyme might contribute to the decomposition of konjac glucomannan in human digestive tract.

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The effects of various plant growth regulators and nutrients on cell growth and anthraquinone production in cell suspension cultures of Rubia akane NAKAI were investigated. Use of an optimized medium resulted in a two-fold increase in the anthraquinone content. From the cell cultures 1, 2-dihydroxyanthraquinone was isolated as the main constituent.

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A neuropeptide named pheromone biosynthesis activating neuro-peptide (PBAN) stimulates the pheromone production of lepidopteran insects. We have identified a different type of cDNA for PBAN, which shows two amino acid replacements in the region of the PBAN sequence previously reported. Single strand conformation polymorphism (SSCP) analysis revealed that the two types of cDNA originated from two allelic variants of the gene for PBAN.

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The levels of endogenous GAs in the anthers of rice (Oryza sativa L., cv. Reimei (normal)) and in those of sterile and fertile plants of a thermosensitive genetic male sterile line (Norin PL12; derived from Reimei) were measured by enzyme-linked immunosorbent assay (ELISA). High levels of GA_4/7 were detected in the anthers of Reimei (normal fertility) and fertile (growing under fertile conditions) PL12, while such levels were markedly reduced with increased sterility in PL12. The anthers of the plants exposed to the sterile conditions contained unldeveloped pollens. These results suggest that the occurrence of GA_4/7 was closely related to the expression of the sterility gene and the growth and/or development of the anther and/or Pollen PL12.

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We addressed the strategies of mapping protein binding sites on a DNA fragment by atomic force microscopy (AFM). The protein binding site was uniquely mapped by distinguishing two termini of a linear DNA fragment. Our simple methods were found very useful to get information on transcription regulatory regions by taking advantage of long range and quick scanning by AFM.

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Four prolyl endopeptidase inhibitors isolated from actinomycetes, named propeptin, SNA-8073-B, staurosporine, and enduracidin were classified into 3 groups on the basis of their inhibition potency against prolyl endopeptidase from a bacterium (Flavobacterium) and a mammal (human placenta). Staurosporine inhibited the enzyme from Flavobacterium more strongly than that from human placenta. Enduracidin inhibited the enzyme from human placenta more strongly than that from Flavobacterium. Propeptin and SNA-8073-B, both new compounds, inhibited the enzymes from both origins to the same extent.

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A marine, obligate anaerobic bacterium, SS71, isolated from a coastal sediment, was a Gram-positive, asporogenous, rod-shaped organism with a G + C mol% of 37.3±0.1. This bacterium produced an α-amylase with a molecular mass of 91 kDa and an isoelectric point of 4.3. The α-amylase had an optimal pH of 7.0 and an optimal temperature of 35°C. The enzyme activity was promoted by 0.5-2.0% NaCl.

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The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum lysine- and threonine-producing mutants were cloned, using the polymerase chain reaction, and sequenced. We found the amino acid substitutions, Val104Ile in the lysine-producing mutants in which HD may cause leaky mutation and Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.

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The metabolism of gibberelline (GAs) in intact plants of Raphanus sativus was investigated. With [²H]GA feeds, [²H]GA₁ from [²H]GA₄, and [²H]GA₄ and [²H]GA₂₀ from [²H]GA₉ were metabolized. Since [²H]GA₂₀ was not converted into [²H]GA₁, endogenous GA₁ may have been biosynthesized from GA₉ via GA₄ rather than from GA₂₀. The radioactivity of [³H]GA₉ and [³H]GA₂₀ was much more strongly transported among the plant organs than that of [³H]GA₁ and [³H]GA₄.

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We consructed two new T-vectors called pUCTA119 and pUCTA18, derived from pUC18. The vectors were designed to produce single thymidine (T)-overhangs when digested with a restriction enzyme Eam1105I. The use of the vectors provides a very rapid system for direct TA cloning and subsequent sequencing of unmodified PCR products since Taq DNA polymerase preferentially adds an adenosine (A) residue to the 3' end of the products under standard PCR conditions.

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Brain ischemia injury is elicited by the excitotoxicity of L-glutamate. Carquinostatin B was isolated from Streptomyces exfoliatus 2419-SVT2 as a potent neuroprotective substance which protests neuronal hybridoma N18-RE-105 cells from L-glutamate toxicity. The structure of carquinostatin B was established principally by NMR studies to be a carbazole derivative with an ortho quinone function.

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Respiratory oxygen uptake was not suppressed by the inhibitor of the quinol oxidation site of cytochrome b/f complex 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), but by farred illumination in Synechocystis PCC 6803 cells grown photoautotrophically. It is proposed that cytochrome b/f complex is not involved in the respiratory electron transport.

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In order to develop naturally occurring antioxidants from edible plants, the antioxidative effect of hot water extracts of Lagerstroemia speciosa leaves, known by the Tagalog name of banaba in the Phillipines, was studied. The content of tannin in banaba extract was 36.8%, in dry weight. Banaba extract showed strong antioxidative activity in a linoleic acid autoxidation system. Banaba extract was found to have a potent radical scavenging action on 1, 1Ldiphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide radicals (O^2-) generated by a hypoxanthine (HPX)/xanthine oxidase (XOD) system. In vitro lipid peroxidation of rat liver homogenate induced by tert-butyl hydroperoxide (BHP) was inhibited by the addition of banaba extract in a dose-dependent manner. From these results, banaba extract was demonstrated to be useful as an anti-oxidant or free radical scavenger to protect biological systems against oxidative stress.

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A rapid HPLC method with fluorescence detection of yessotoxin (YTX) and its two analogs (45-OHYTX and norYTX) in mussels and scallops is presented. A dienophile reagent, DMEQ-TAD, was used for fluorescence labeling. YTX was measured in the range 1-100 ng. The method confirmed the occurrence of YTX and 45-OHYTX for the first time in mussels from Chile and New Zealand.

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We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40°C. It was stable up to 60°C at pH 6, and was inhibited by Hg²⁺ and p-quinone (K_i=0.18 mM). The molecular weight of the enzyme was about 140, 000, and those of the subunits were 69, 000, 51, 000, and 20, 000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The α-helix content was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of D-mannitol with an apparent K_m of 98μM (for D-mannitol) and V_max of 213 μmol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.

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The absolute configuration of pectenotoxin-6 (4, PTX6), found in association with diarrhetic shellfish poisoning, was identified by NMR spectroscopy using a chiral anisotropic reagent, phenylglycine methyl ester (PGME), which was condensed with a carboxyl group of 4.

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Staphylococcal γ-hemolysin consists of HγI (or LukF) of 34 kDa and HγII of 32 kDa, which cooperatively lyse human erythrocytes. Our previous data showed that the N-terminal 57-residue segment of HγII is the essential region for the HγII function [H. Nariya and Y. Kamio, Biosci. Biotech, Biochem., 59, 1603-1604 (1995)]. To identify the minimum amino acid residues in the 57-residue segment responsible for the specific hemolytic activity, a series of mutant genes were constructed and expressed in Escherichia coli. The mutant proteins were purified and assayed for their hemolytic activity. The results indicate that the 5-residue segment (K²³R²⁴L²⁵A²⁶I²⁷) of HγII is the minimum region essential for the HγII function.

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An open reading frame was found 214 bp downstream of the cellulose synthase operon of Acetobacter. The encoded amino acid sequence was found to be similar to some beta-glucosidases (G3ases). We detected G3ase activity in the culture medium and analysis of the N-terminal amino acid sequence showed that this gene encodes the enzyme. Therefore, it is possible that this region is a gene cluster for cellulose synthesis.

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