Amino acid analogues are of considerable interest as inhibitors of enzymes involved in amino acid and peptide metabolism. In particular, α-aminoalkylphosphonic acids and 2-aminoalkylboronic acids, in which the carboxyl group of amino acids is replaced by a phosphonic acid or boronic acid function, respectively, constitute a unique class of amino acid mimics from which a number of potent enzyme inhibitors have been prepared. The inhibitory activity mainly stems from the fact that the tetrahedral phosphonic moiety or the tetrahedral adduct of electrophilic boronic acid is a good mimic of the putative tetrahedral transition state or intermediate encountered in the enzymatic hydrolysis or formation of peptides. Since the peptide hydrolysis and formation invariably involves the tetrahedral high energy species in the course of the reaction, these amino acid mimics serve as a general key element for inhibitors of a broad spectrum of proteases and peptide ligases. The transition state analogy of aminophosphonic- and aminoboronic acid-derived inhibitors also gives a clue to the detailed reaction mechanisms of the enzymes by X-ray crystallographic and NMR analysis of the enzyme-inhibitor complex.

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Some Acetobacter strains produce a cellulose called bacterial cellulose in culture medium. This cellulose has unique structural and mechanical properties compared with higher plant cellulose, and is expected to be a commodity material in various fields of industry. For economical mass production, it is essential to construct an aeration and agitation culture process. To explore the industrial applications of the bacterial cellulose, the structural features and physicochemical properties need to be understood and potentially improved. This paper reviews recent progress in research on this bacterial cellulose.

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The biological effect of antioxidants which showed high superoxide-scavenging (SOS) activity in an in vitro analysis was examined by using Drosophila melanogaster. When the flies were exposed to paraquat as an endogenous source of the superoxide anion, their survival rapidly decreased, Although the SOS antioxidants did not have a preventive effect against paraquat toxicity, a supplement of such SOS antioxidants as glutathione, (+)-catechin and/or (-)-epicatechin to the diet had a reparative effect on flies damaged by the superoxide anion. The survival ratio of flies fed on a diet enriched with SOS antioxidants ranged from 77% to 87%, while that of the control group was 56%. When flies were exposed to paraquat in the presence of hydrogen peroxide or iron, each combination was more toxic than paraquat alone, since the two compounds could accelerate the generation of reactive oxygen species in vivo. The SOS antioxidants, however, allowed the flies to resist the combined toxicity of paraquat and ferrous iron.

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The effects of a low-casein diet fortified with methionine and threonine on renal cortical and glomerular transforming growth factor (TGF)-β activity were studied in rats with nephritis induced by anti-rat kidney glomerular basement membrane antiserum. Both normal and nephritic rats were fed experimental diets for 10 days. An injection of nephrotoxic serum increased urinary protein excretion and renal TGF-β activity. A methionine-threonine-supplemented 8.5% casein diet, compared with a basal 20% casein diet, decreased these two measurements without aggravating growth retardation in nephritic rats. These results suggest that aggravation and alleviation of symptoms incident to anti-GBM nephritis are relevant to elevation and reduction of TGF-β activity, respectively. The results also suggest that amino acid-balanced low-protein diets would have beneficial effects on glomerulonephritis without causing severe protein malnutrition.

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The effects of tropomyosin (TM) on the stability of actin with and without heavy meromyosin (HMM) during storage at low temperature were studied by the DNase I inhibition assay. The stabilizing effect of TM on actin without ATP at 0°C was associated with the extent of binding of TM to the actin filaments that was dependent on the KCl concentrations: i.e., TM effectively depressed the denaturation of actin at a KCl concentration ranging from 0.1 to 0.5 M whereby TM can be bound to F-actin, Stabilization by TM could reduce the probability of the destabilization of actin due to a small amount of HMM. A combination of TM and a large amount of HMM strongly stabilized actin, even in a high salt concentration such as 0.6 M KCl These results indicate that tropomyosin played an important role in stabilizing actin in an actomyosin complex (natural actomyosin) during treatment with salt at a low temperature.

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In the previous study, we proposed that phosphoryl oligosaccharides (POs) prepared from potato starch had an inhibitory effect on the formation of a calcium phosphate precipitate in vitro. In this study, we investigated the structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that was the main component of POs. By treating with bacterial saccharifying α-amylase (BSA) after glucoamylase (GA), the PO-1 fraction produced 3²-phosphoryl maltotriose from 3-phosphoryl oligosaccharides, and 6³-phosphoryl maltotriose from (6-phosphoryl oligosaccharides. These products were characterized spectrometrically as well as chemically, including measurement of the amounts of the non-reducing-terminal residue, reducing-terminal residue, and organic phosphate. A small amount of 6²-phosphoryl maltose was also detected after treating with GA alone, indicating that 6²-phosphoryl maltotriose existed in the PO-1 fraction. According to the reaction specificities of GA and BSA, we conclude that the PO-1 fraction was made up of 3-phosphoryl oligosaccharides (3³-phosphoryl maltopentaose and 3⁴-phosphoryl maltopentaose) and 6-phosphoryl oligosaccharides (6³-phosphoryl maltotriose, 6²-phosphoryl maltotriose, 6³-phosphoryl maltotetraose, and 6⁴-phosphoryl maltopentaose).

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The egl2 gene encoding a thermostable endoglucanase (EGL2) was cloned from Humicola grisea. The DNA sequence of egl2 predicted two putative introns in the coding region. The deduced amino acid sequence of EGL2 was 388 amino acids in length and showed 99.5% identity with the H. insolens CMC 3. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5' upstream region of the egl2 gene. The egl2 gene was expressed in Aspergillus oryzae, and EGL2 was purified. EGL2 produced by A. oryzae showed a high activity toward carboxymethyl cellulose. The optimal temperature of EGL2 was 75°C, and EGL2 had more than 80% residual activity after heating up to 75°C for 10 min. This is the first report of enzymatic properties of the EGL2-type thermostable cellulase homologs from Humicola.

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A guar gum-degrading enzyme of the newly isolated Bacillus circulans K-1 was purified to an electrophoretically homogeneous state. The molecular weight of the purified enzyme was 62, 000 by SDS-PAGE. The purified enzyme was separated into at least six isozymes by isoelectric focusing and the pI of these isozymes were 5.4, 5.5, 5.6, 5.8, 6.0, and 6.2, respectively. The N-terminal amino acid sequences of the typical three of these proteins were all the same, Ala-Ser-Gly-Phe-Tyr-Val-Ser-Gly-Thr-Lys-Leu-Leu-Asp-Ala-Thr-Gly-Gln-Pro-Phe-Val-Met-Arg. The enzyme was most active at pH 6.9 and at 64°C. The enzyme was activated slightly by Al³⁺ and inhibited strongly by Sn²⁺ and Zn²⁺, N-bromosuccinimide, 2-mer-captoethanol, and ethylenediamine-tetraacetic acid.

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We describe here different regulation of the AldP gene, a nuclear gene encoding chloroplast aldolase, in different tissues and growth ages of rice seedlings. Expression of the AldP gene is mesophyll cell-specific, and increases from the basal to the upper region in each leaf. The gene expression is repressed in the dark-grown leaf blade, but is induced by a short-term-exposure to light, to a level higher than that seen in the normal leaf blade. However, the light-inducibility differs among the tissues, and shows different patterns among leaf positions; i.e., the extent of light-induction is higher in the third leaf blade as compared with the earlier developed second leaf blade. Such positional differences in the regulation are also seen in the leaf sheath. These responses are not accompanied by changes of the cell type specificity in the expression.

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Many antioxidants have been found in spices and herbs, and some of them are well known as strong scavengers of active oxygen radicals. We have isolated active products, which markedly inhibited the formation of malondialdehyde (MDA) from 2-deoxyribose and the hydroxylation of benzoate with the hydroxyl radical, from methanol extracts of allspice and clove. Pimentol from allspice, and biflorin and its isomer, abbreviated as clove3, from clove were identified as the active principles. These revealed strong activity as hydroxyl radical scavengers at a concentration of 2.0μM. The antioxidative activities in an in vitro model system involving the rabbit erythrocyte membrane ghost were as strong as those of α-tocopherol at 200 μM. Such advanced glycation end products (AGE) as pentosidine are biomarkers of diabetes mellitus, and active oxygens have been suggested to be involved in the formation of AGE. The above-mentioned free radical scavengers effectively inhibited the formation of pentosidine in a model system of N^α-t-butoxycarbonyl-fructoselysine and N^α-t-butoxycarbonyl-arginine.

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Polysaccharide that had been extracted with 1% sodium carbonate from Rooibos leaves (Aspalathus linearis) showed strong anti-HIV activity. Du-Zhong leaves also showed anti-HIV activity, although lower than the extract of Aspalathus linearis, but Japanese tea leaves and a hot water extract of Aspalathus linearis did not. The anti-HIV activity of the alkaline extract from Aspalathus linearis was recovered mainly in the 25-75% ethanol-precipitated fraction. The polysaccharide almost completely inhibited the binding of HIV-1 to MT-4 cells, It is inferred from these results that the polysaccharide from Aspalathus linearis is involved in the mechanism for virus binding to T cells.

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Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II). The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II. The Pu-NPase I gene of TH 6-2 has been cloned, sequenced, and expressed in E. coli. The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29, 637. The deduced amino terminus sequence completely coincided with that found for the purified enzyme. The cloned gene was overexpressed in E. coli by using the trc promoter to produce an active enzyme in large quantities. The amino acid sequence of Pu-NPase I shared 50%, similarity with those of human and mouse purine nucleoside phosphorylases.

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Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPases). The type I enzyme (Pu-NPase I) is a functional and structural homolog of eukaryotic purine nucleoside phosphorylases that catalyze the phosphorolysis of inosine, guanosine, and their derivatives. The type II enzyme (Pu-NPase II) is a minor enzyme that efficiently phosphorolyzes adenosine and its derivatives rather than other purine nucleosides like Escherichia coli Pu-NPase. The gene coding for Pu-NPase II (punBgene) has been cloned and sequenced from TH 6-2 strain. The deduced amino acid sequence of Pu-NPasce II shared 54% identity with that of E. coli enzyme, while it had no significant homology to that of Pu-NPase I or eukaryotic enzymes. By producing the Pu-NPase II in E. coli cells, the Pu-NPase II has been purified and characterized.

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The oxidative stability of ethyl linoleate (LA), ethyl linolenate (LN), and ethyl docosahexaenoate (DHA) in an aqueous solution was compared by measuring the decrease in unoxidized substrate and the formation of total hydroperoxides, conjugated dienes, and monohydroperoxides (MHPs). The highest stability was shown by DHA, this being followed by LN and LA in both cases when using Fe²⁺-ascorbic acid and 2, 2'-azobis(2, 4-dimethylvaleronitrile) (AMVN) as a initiator, while the reverse order of oxidative stability was obtained when the esters were oxidized in chloroform or ethanol with AMVN. The stability of MHPs in an aqueous solution also increased with increasing degree of unsaturation. HPLC analyses showed little difference in the positional distribution of MHP isomers between aqueous oxidation and autoxidation in the air. This result suggests no selective abstraction of hydrogen atoms from the bisallylic positions of polyunsaturated fatty acid in an aqueous phase. The product ratio of hydoroperoxy epidioxides to MHPs in the aqueous oxidation of LN was lower than that of autoxidized LN in the air, showing the rapid decomposition of epidioxides in an aqueous solution.

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Apolipoprotein (apo) A-1 cDNA was amplified by the reverse-transcriptase-polymerase chain reaction (RT-PCR). Primers were synthesized according to the nucleotide sequence of chicken apo A-1, and the identity of apo A-1 cDNA was confirmed by comparing with the N-terminal amino acid sequence. The open reading frame of apo A-1 cDNA consists of 795 nucleotides, and it is capable of coding a polypeptide of 264 amino acids. A comparison between quail and chicken apo A-1 revealed 94.5% homology in the nucleotide sequence and 91.7% homology in the amino acid sequence. There was a similar 11- or 22-amino acid repeat in quail apo A-1 as was the case for chicken apo A-1. Apo A-1 mRNA was evaluated to be 1.4 k in length and was expressed in various tissues of Japanese quail: the liver, small intestine, lung, kidney, heart, and muscle. A quantitative evaluation, however, revealed that the liver and small intestine were the major organs for apo A-1 synthesis, accounting for more than 90% of the total expression of apo A-1 mRNA. Besides apo A-1 mRNA (1.4k in length), a transcript of 4.1 k was detected in all the tissues examined, with a magnitude ranging from 5 to 10% of the apo A-1 mRNA level. The effect of cholesterol level on the expression of apo A-l mRNA was studied to address the physiological significance of apo A-1 in the liver, small intestine, and muscle. The level of cholesterol in the liver and breast muscle was increased by feeding with cholesterol and reached a saturation level at day 7. There was also a temporal rise of cholesterol level at day 7 in the small intestine. Dietary cholesterol increased the expression of apo A-1 mRNA two fold in both the liver and small intestine. This was not the case for breast muscle, in which the expression of apo A-1 mRNA was not modulated by the cholesterol level.

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Ricin D, one of two isolectins from small castor beans showed stronger cytotoxicity than another one, ricin E, based on the inhibition of colony formation and the inhibition of protein synthesis. Both ricin D and ricin E induced cell lysis to different extents in each cell line tested, albeit ricin E was slightly less effective than ricin D. DNA fragmentation, a characteristic feature of apoptosis, was also induced by ricin D and ricin E in Vero cells. Scatchard plot analysis showed that ricin D binds to celles with higher affinity than ricin E, while the number of binding sites per cell was not much different, suggesting that the differences in the cytotoxicity between ricin D and ricin E is mainly due to their differential binding affinity to cells. In Vero cells, the cytolytic activities of ricin D and ricin E were inhibited by brefeldin A (BFA), which is known to affect the Golgi apparatus, but no significant effect of BFA was observed in a BFA-resistant cell line, MDCK cells. These results suggest that the Golgi apparatus may be involved in ricin-induced cell lysis.

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An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis showed a putative promoter region, a putative transciptional termination signal, and an open reading frame of 963 bases, coding for a polypeptide of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl 650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino acids) of the purified protein was coincident with Asp16-Va145 of the deduced amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region were removed during the purification procedures. The deduced amino acid sequence showed high similarity with microbial intracellular serine proteases. The molecular mass of this enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme was stable at pH 6.0-12.0 and below 60°C in the presence of Ca²⁺. The temperature and pH optima of the enzyme were 65°C and pH 11.0, respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA and EDTA.

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5-Deoxy-5-oxomilbemycin A₄ 5-hydrazone derivatives were synthesized by a condensation reaction of 5-deoxy-5-oxomilbemycin A₄ (2) with hydrazines. Acetic acid played an important role to give each hydrazone in a good yield by regulating the reactivity of ketone 2 and the hydrazines. The acaricidal activity of each synthesized compound was studied on the primary leaves of plants of the Vigna sinensis Savi species infected with organic phosphate-sensitive mites (Tetranychus urticae). Some of the synthesized derivatives exhibited higher miticidal activity than that of milbemycin A₄. Among them, 5-deoxy-5-oxomilbemycin A₄ 5-N-(N', N'-dimethylcarbamoyl)hydiazone (18) totally controlled the mites at a concentration of 3 ppm.

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The soluble and insoluble fractions obtained after sonication and centrifugation of Bifidobacterium adolescentis M101-4 cells were examined, and both of these fractions exhibited mitogenic activity in an assay of murine splenocytes and Peyer's patch cells in vitro. The soluble fraction was further treated by a 6-step procedure involving proteinase K-treatment, ultrafiltration with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography, dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and gel-filtration to yield a soluble high molecular weight fraction (SHF) which was effective for stimulating the proliferation of murine splenocytes. Almost three quarters of this fraction by weight was found to consist of carbohydrates containing glucose and galactose as major constituents, and the average molecular weight was estimated to be between 60, 000 and 2, 460, 000, with the main peak at 1, 550, 000 Da, by the retention time of gel permeation chromatography. A structural analysis by ¹H- and ¹³C-nuclear magnetic resonance and methylation indicated that SHF contained polysaccharides consisting of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf1- and -6Galf1- as the minor residues. Immunopotentiating SHF was found to contain galactofuranosyl residues as characteristic constituents which had not been previously detected in other soluble fractions from Gram-positive bacteria.

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Several strains of acetic acid bacteria belonging to the genus Acetobacter, showing strong acetate oxidation, were screened and their microbiological aspects in acetate oxidation were investigated. When all available carbon and energy sources were exhausted and only acetic acid remained in the late stationary phase, the bacteria started to consume the acetic acid that had been accumulated in the culture medium for vinegar fermentation. They grew rapidly, showing the second stationary phase and a typical biphasic growth curve was observed. The cells from the first growth phase were acid tolerant, while the cells from the second growth phase turned over to become acid sensitive. However, no distinct acetate oxidation took place when oxidizable ethanol and other available carbon sources still remained in the culture medium. Moreover, no apparent acetate oxidation was observed in vinegar mash in which more than 4.5% of acetic acid was allowed to accumulate. There was a threshold in acetate concentration since the most selected strains oxidized acetate when the final concentration of acetic acid accumulated was less than 3.7%. When only acetic acid was administrated as the sole carbon and energy sources, the organisms finally used acetic acid after a long lag time. The lag time was shortened by the addition of a small amount of readily usable energy source, such as ethanol. From enzymatic analysis, only acetyl-CoA synthetase increased much among the enzymes concerning acetyl-CoA formation from acetate, while the enzyme activities of acetate kinase and phosphotransacetylase were not changed significantly. The enzyme activities or isocitrate lyase and malate synthase also increased significantly in the cells when acetate was consumed. These results indicate that acetic acid is converted to acetyl-CoA by acetyl-CoA synthetase to put acetate into the TCA cycle as well as to the glyoxylate cycle allowing the bacteria to grow rapidly on acetic acid after ethanol exhaustion. Taking together with growth experiments and enzymatic data accumulated, it was strongly suggested that cells different in physiological characteristics from the first growth phase emerged in the second growth phase.

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Gypsophila elegans contains a new type 1 ribosome-inactivating protein, which we named gypsophilin. The protein was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation, ion-exchange chromatography, and adsorption chromatography. The protein was found to have a molecular mass of 28.0 kDa and a pI of about 10.1. It does not contain glycosidic linkages. The sequence of the N-terminal 22 amino acids of the protein shows a close relationship to other RIPs. The enzyme strongly inhibits protein synthesis in a rabbit reticulocyte lysate and depurinates 28S rRNA in rat liver ribosomes in a manner identical to that of ricin A-chain and other RIPs. Using a direct method for the measurement of the RNA N-glycosidase activity, the substrate spcificity of gypsophilin was identified. EC₅₀ of the protein for ribosomes of rat liver, wheat germ, and E. coli was 39.8 pM; 0.24 nM, and 0.82 μM, respectively. Gypsophilin may be one of the most active RNA N-glycosidases among the RIPs known to date. Immunoelectron microscopic localization of gypsophilin in the leaves shows that the protein is accumulated densely in the intercellular spaces and is also distributed within vacuoles in the cytoplasm.

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The inhibitory effect of hop bract polyphenols (HBP) on cariogenic streptococci was investigated. It was found that the high molecular weight polyphenol (estimated about 36, 000-40, 000) inhibited the cellular adherence of Streptococcus mutans MT8148 (serotype C) and Streptococcus sobrinus ATCC 33478 (serotype g) at much smaller concentrations than the polyphenols extracted from oolong tea or green tea leaves. Furthermore, HBP also inhibited the action of glucosyltransferase, which was involved in the water-insoluble glucan synthesis, but did not suppress the growth and the acid production of the bacteria. These results suggest that HBP would be a candidate to act against dental caries caused by Mutans Streptococci.

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This study investigated ectoenzyme release from small intestine brush border membranes (duodenum and jejunum, Preparation A; ileum, Preparation B) of mice by the action of phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D. Most of the alkaline phosphatase was solubilized from Preparation A, but about 60% was released from Preparation B. As for alkaline phosphodiesterase I activity, 15 and 10% were released from Preparations A and B, respectively. With Preparation B, octylglucoside treatment followed by phosphatidylinositol-specific phospholipase C or glycosyl-phosphatidylinositol-specific phospholipase D completely solubilized the alkaline phosphatase activity. However, this treatment did not change the ratio of release of alkaline phosphodiesterase I from Preparation A or B. These results indicate that the resistance to alkaline phosphatase found in Preparation B is due to hindered accessibility of the bonding splitting by phosphatidylinositol-specific phospholipase C and not to a modified glycosyl-phosphatidylinositolanchor.

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Polyclonal antibodies against glutaraldehyde-treated rabbit serum albumin (pRSA) and human serum albumin (pHSA) were prepared from rabbit and mouse, respectively. Anti-pRSA antibody had the structural determinant depending on the polymerization process of RSA and showed only a weak cross-reactivity with the other glutaraldehyde-treated albumins. Anti-pHSA antibody (after adsorption of anti-HSA antibody) recognized only pHSA, but not HSA and the other treated albumins. The cross-reactivity of those antibodies was examined with albumins treated by other methods such as modification of glucose and fructose, carbodiimide, and transglutaminase. Among them, RSA and HSA modified with glucose and fructose had an affinity for each antibody and the reactivity depended on the extent of formation of the polymerized albumin. The result suggests that functional groups involved in cross-linking of albumin are important for formation of the cross-reactivity with the antibody and that a definite structure immunochemically similar to glutaraldehyde-treated albumin could be formed by the Maillard reaction.

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The effects of olive fruit extract on arachidonic acid lipoxygenase activities were investigated using rat platelets and rat polymorphonuclear leukocytes (PMNL). Olive extract strongly inhibited both 12-lipoxygenase (12-LO) and 5-lipoxygenase (5-LO) activities. One of the compounds responsible for this inhibition was purified and identified as 2-(3, 4-dihydroxyphenyl)ethanol (DPE). DPE inhibited platelet 12-LO activity (IC₅₀, 4.2μM) and PMNL 5-LO activity (IC₅₀, 13μM) but not cyclooxygenase activity in cell-free conditions. It also inhibited 12-LO activity in intact platelets (IC₅₀, 50μM) and reduced leukotriene B₄ production in intact PMNL stimulated by A23187 (IC₅₀, 26μM). The inhibition by DPE of both lipoxygenase activities was stronger than that by oleuropein, caffeic acid, or 7 other related phenolic compounds, especially in intact cells. These results suggest that DPE is a potent specific inhibitor of lipokygenase activities.

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Component sphingoid bases of cerebrosides from leaves of twenty-two plants were analyzed by capillary GC as the corresponding fatty aldehydes. No apparent difference in the ratios of 8-E-unsaturated sphingoid bases to the 8-Z-forms was found between chilling-resistant and chilling-sensitive plants. Nevertheless, most of the chilling-resistant plants analyzed had more 8-Z-unsaturated trihydroxy base contents than those of the E-isomer.

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The serum cholesterol level in rats was significantly decreased in a group fed on a soyprotein peptic hydrolyzate (SPH) when compared with a group fed on a casein tryptic hydrolyzate (CTH). The fecal excretion of total steroids was significantly greater with rats fed on the SPH diet when compared with the CTH diet. The results of CaCo-2 studies clearly suggest that the suppression of cholesterol absorption in the intestinal epithelia is part of the mechanism for the hypocholesterolemic action induced by SPH.

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Dabsyl-alginate, the dye-labeled substrate of alginate lyase, was prepared. Low-viscosity alginate, which was prepared by enzymatic degradation of sodium alginate, and tetramethylenediamine were conjugated by reductive amination. Then, dabsyl-Cl was coupled with the primary amino group of the aminobutyl-alginate. The assay for endo-alginate lyase using dabsyl-alginate was simple to use. The amount of dyed fragment released from the substrate increased with the amount of the enzyme.

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Twenty-nine strains of yeasts, which are capable of using galactose, melibiose, or raffinose, were screened for α-galactosidase production. Among the strains, 5 produced intracellular and extracellular α-galactosidases, and 2 produced only intracellular enzyme. Substrate specificities of these enzymes were explored using 6³-α-D-galactosyl-1, 4-β-D-mannotriose and 6³-α-D-galactosyl-1, 4-β-D-mannotetraose. All enzymes liberated the terminal galactose from 6³-α-D-galactosyl-1, 4-β-D-mannotriose, but did not the stub galactose from 6³-α-D-galactosyl-1, 4-β-D-mannotetraose.

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Clones of a gene encoding an endo-1, 4-β-glucanase (EC 3.2.1.4) were obtained from Bacillus sp. 22-28, and the nucleotides were sequenced. A recombinant plasmid, pMK5, included a complete ORF of 2352bp that encoded 783 amino acid residues. Another plasmid, pM3, which showed enzymatic activity in E. coli JM109, was also obtained, and it included an incomplete ORF of 1873bp, which lacked the original stop codon and 479 bp of the C-terminal region. The enzymes purified from both of the two types of transformants have shown almost the same properties in comparison with that of the wild type Bacillus sp. 22-28.

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We investigated the possible effects of acetic acid bacterium on ethanol oxidation in vivo by monitoring the blood ethanol level after injecting 5% ethanol with (treated group) of without (control group) a freeze-dried bacterial cell suspension directly into the stomach of anesthesized rats. Paired comparison t-tests of the results indicate that the blood ethanol concentration of the rats in the treated group was significantly (p < 0.07) lower than that in the control group. When measured 10 min after administering ethanol into the gullet, the concentration in the stomach of the rats that received acetic acid bacterium simultaneously with ethanol was significantly (p < 0.10) lower than that of the rats that received ethanol alone. We consider that freeze-dried cells of acetic acid bacterium oxidized ethanol in the stomach and could be effective for reducing the blood ethanol level after drinking.

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The quality and quantity of flavonoids from two types of propolis collected by two different varieties of Apis mellifera bee in the same region were investigated. There was found a remarkable quantitative and qualitative difference of flavonoids in propolis. These results indicate that the chemical composition of propolis was dependent on the variety of the bee.

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We found that daidzein stimulated bone resorption in the pit formation assay at the concentration of 10^-8-10^<-10&t; M. On the other hand, genistein and ipriflavone at these concentrations did not have any effect on pit formation. We speculated that daidzein had the ability to induce osteoclasts directly or indirectly from their progenitors and might be a tool to study osteoclast differentiation.

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In addition to the two-component regulatory system, DegS-DegU, five other genes degR, degQ, senS, tenA, and proB positively regulate the production of Bacillus subtilis exoproteases when they are present on a multicopy plasmid. To study the extent of involvement of these genes in exoprotease production in a single copy state and possible relationship among these genes, strains carrying single or multiple disruption mutations in these loci were constructed, and the expression of aprE'-'lacZ was analyzed. From such studies, the following results were obtained with respect to the regulation of aprE expression. i) Disruption mutations were divided into two groups; one (degQ and degR) significantly reduced aprE expression, while the other (senS, tenA, and proB) had little effect on it. ii) SenS was involved in temporal regulation. iii) The combined effects of dome of the disruption mutations tested were not necessarily additive. iv) The extent of the negative effects generated by the mutations depended on the medium used.

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One of the enzymes from bakers' yeast that catalyzes the reduction of α- and β-keto esters has been studied. The N-terminal amino acid sequence of the enzyme shows that the enzyme belongs to the aldo-keto reductase superfamily. The substrate specificity of the enzyme is broad and resembles those of other aldo-keto reductases. The enzyme catalyzes the reduction of keto esters, aldehydes, and aldohexoses.

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Homooligomycin E (1) was isolated from the culture broth of Streptomyces ostreogriseus and its structure was analyzed on the basis of physicochemical and spectroscopic data. It showed strong cytotoxicity against several human tumor cell lines.

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In order to investigate the specificity of peptide bond cleavage by acid proteinase A from Aspergillus niger var. macrosporus (Aspergillopepsin II), performic acid-oxildized bovine pancreatic ribonuclease A was digested by the enzyme at pH 1.8 or 5.5, and the resulting peptides were separated by HPLC and analyzed. Among the total 123 peptide bonds, approximately thirty and thirteen bonds were cleaved at pH 1.8 for 2 h and at pH 5.5 for 20 h, respectively. Cleavages occurred fairly specifically at Tyr-X, Phe-X, His-X, Asn-X, Asp-X, Gln-X, and Glu-X bonds.

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An NADP-malic enzyme [EC 1.1.1.40] was purified to homogeneity from Bradyrhizobium japonicum A1017, and the molecular and physiological characteristics were surveyed. The molecular mass of one subunit of the purified enzyme was evaluated to be 77, 600 Da by SDS-PAGE, and the native enzyme was a tetramer in pH 7.0 and dimer in pH 8.0 conditions, showing complex oligomeric characteristics corresponding to pH value.

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Inhibitors of acid phosphatase were found in soybeans. They were extracted with hot-water, and partially separated from the other soybean components by alcohol precipitation and ion-exchange chromatography. They seemed to be a kind of acidic polysaccharide.

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Mutations were introduced into the reactive-site region of human pancreatic secretory trypsin inhibitor (PSTI) to produce thrombin and/or factor Xa inhibitors. All of five mutants showed trypsin inhibitory activity as strong as wild-type PSTI. Moreover, the Arg (P1), Pro-Arg (P2-P1), and Pro-Arg-Ile-Tyr-Asn (P2-P1-P1'-P2'-P3') (bold letters indicate replaced amino acids compared to the wild type) mutants had additional inhibitory activities toward factor Xa, both thrombin and factor Xa, and thrombin, respectively, at 1×10^-5M.

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A model tripeptide, Gly-L-Leu-L-Phe, was immobilized with activated aminomethyl polystyrene, and its C-terminal was reduced to an alcohol. This peptidyl alcohol was selectively hydrolyzed at the C-terminal amide bond to afford a polymer-supported dipeptide (Gly-L-Leu) and amino alcohol (Phe-OH). The amino alcohol, including it's absolute configuration, was determined by labelling with (+)-MNB-COOH, and the dipeptide was reused for a determination of its C-terminal amino acids. The D, L-amino acids of the tipeptide were sequentially determined from the C-terminus.

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Mitochondria were isolated from Hansenula anomala induced for cyanide-resistant respiration by antimycin A-treatment. Cyanide-resistant respiratory activity in isolated mitochondria was stimulated by AMP, ADP, dAMP, and GMP, but not by ATP, adenosine, cAMP, 2'(3')-AMP, CMP, and UMP. Effects of nucleotides were also observed on cyanide-resistant and salicylhydroxamate-sensitive decylubiquinol oxidase activity. Carbonylcyanide m-chlorophenylhydrazone, oligomycin, and carboxyatractyloside did not affect activation of decylubiquinol oxidase activity by AMP. It is suggested that purine nucleoside 5'-monophosphate or diphosphate stimulates alternative oxidase activity from the outer surface of the mitochondrial inner membrane with a mechanism different from respiratory control. Alternative oxidase activity might be controlled by adenine nucleotides posttranscriptionally in fungi.

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Prodigiosin 25-C and metacycloprodigiosin were found to suppress PTH-stimulated pit formation by cultured osteoclasts on bone slices. They also inhibited the acidification of vacuolar organelles in intact osteoclastic cells. Since the acidic pH in these organelles is generated by the action of proton-pumping ATPases of the organelle, these results indicate that the proton-pumping activity of V-ATPase in osteoclastic cells is essential in bone resorption and that the inhibition of the acidification of vacuolar organelles by prodigiosins results in suppression of PTH-stimulated bone resorption.

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