Staphylococcus aureus is an important opportunistic pathogen. It produces a variety of extracellular proteins, which may play important roles in infections by this bacterium. Staphyloccal α-toxin is a pore-forming 33-kDa polypeptide. In the first part of this article, we will refer to the roles of cell membranes in the pore formation by α-toxin as well as the molecular dissection of α-toxin for understanding its pore-forming nature. Staphylococcal γ-hemolysin and leukocidin are bi-component cytolysins, which have different cell specificities towards erythrocytes and leukocytes, respectively. We have found that these bicomponent cytolysins share a common component. In the second part of this article, we will refer to the current status of knowledge of molecular cloning of the genes coding for γ-hemolysin and leukocidin, molecular domains of the toxins which decide the cell specificities, and mode of action of these bi-component toxins.

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The effects of cholestane 3β, 5α, 6β-triol on intestinal epithelial crypt cells were investigated using the IEC-6 cell line. Cholestane 3β, 5α, 6β-triol decreased SH groups (glutathione and protein SH) in the cell, and showed cytotoxicity in a time-dependent manner. Although the concentration of cholestane 3β, 5α, 6β-triol used in this study (100 μM) was very high compared with that in plasma of experimental animals, cholestane 3β, 5α, 6β-triol did not show any cytotoxicity on IEC-6 cells without fetal calf serum (FCS). The level of cytotoxicity was dependent upon the concentration of FCS in the culture medium. Unknown components in FSC (not VLDL or LDL) were suggested to be associated with the cytotoxicity of cholestane 3β, 5α, 6β-triol. Morbover, the fact that even heat-treated FCS (100°C for 30 min) still mediated the cytotoxicity suggested the participation of non-protein components.

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The ten strains of Issatchenkia species were examined for their partial base sequences of 18S and 26S rRNAs. In the 18S rRNA partial base sequences (positions 1451-1618, 168 bases), the strains of the species of the genus Issatchenkia were found to be not uniform phylogenetically. The calculated base differences numbered 5-0. The strains of Issatchenkia species examined had 3-1 base differences with the type strain of Pichia membranaefaciens. Especially, the type strain of Issatchenkia orientalis, the type species of the genus Issatchenkia was found to be closely related phylogenetically to that of P. membranaefaciens. The calculated number of base differences was only one. The base sequences on the fingerprint segment were comprised of four bases (four kinds of AUAU, CCAU, AUAG, and ACAU), as found in P. membranaefaciens (ACAA). In the 26S rRNA partial base sequences, the calculated number of base differences was 8-0 (positions 1611-1835, 225 bases), and the calculated percent similarities were 61-80 (positions 493-622, 130 bases), within the genus Issatchenkia. Discussion was made phylogenetically and taxonomically, especially on the phylogenetic relationship between the type species of the genera Issatchenkia and Pichia and on a circumscription of the genus Issatchenkia.

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Aspergillus niger, which produces xylan-degrading enzymes, was immobilized on non-woven fabrics. The maximum xylan hydrolysis activity (15U/cm³-support) and the highest stability were obtained when the fungus was immobilized on non-woven fabric made of silk. The enzymatic properties of the immobilized preparation were similar to those of the free enzyme. Ten times repeated batch hydrolysis of birch-wood xylan was done over a period of 450 h. Hydrolysis of different xylan substrates such as oat spelts and rice bran by the immobilized mycelia was also investigated.

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Mutastein, a potent inhibitor of insoluble glucan synthesis by Streptococcus sobrinus, is a protein with a molecular weight of ∼2×10⁶. Amino acid and ELISA analyses suggested that mutastein is a mixture of heterogenous polymers of α-casein contained in the culture medium of the producing strain, Aspergillus terreus M3328. Mutastein strongly inhibited the primer-dependent insoluble glucan synthase of S. sobrinus B13. The primer-independent soluble glucan synthase was not affected by mutastein while primer-dependent soluble glucan synthase was slightly activated.

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A novel α-galactosidase, designated α-galactosidase II, was isolated from the culture filtrate of Mortierella vinacea. The molecular size of the purified enzyme estimated by gel filtration was 60 kDa, which agreed with that, 51-62 kDa, estimated by SDS-PAGE. The enzyme was thermolabile at neutral pH, but the addition of BSA to the enzyme solution at the concentration of 0.01% increased its stability considerably. The enzyme appears to be novel because it showed a distinct substrate specificity from other microbial α-galactosidases on galactomanno-oligosaccharides, prepared from galactomannan, that is, the enzyme liberated not only side-chain 2-galactosyl residue from 6³-mono-α-D-galactopyranosyl-β-1, 4-D-mannotetraose but also terminal α-galactosyl residue from 6³-mono-α-D-galactopyranosyl-β-1, 4-D-man-notriose. In addition, the enzyme acted on galactomannans effectively. α-Galactosidase II cDNA was cloned and its nucleotides sequenced. The deduced amino acid sequence showbd that the mature enzyme consisted of 376 amino acid residues with a molecular mass of 41, 334 Da. The derived amino acid sequence of the enzyme showed 31-49% sequence similarity with those of α-galactosidases from other origins.

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β-Fructofuranosidase [EC 3.2.1.26] in Clostridium perfringens was induced in the presence of sucrose and suppressed in the presence of glucose or maltose. The enzyme seems to be present in protoplasm in a soluble state. The β-fructofuranosidase from C perfringens cells grown on sucrose was purified by ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, and hydroxylapatite chromatography to a homogeneous state. The molecular weight was 37, 000 by gel filtration using Sephadex G-150 and by SDS-polyacrylamide gel electrophoresis. The amino acid composition is not much different from those of other microorganisms, but the Glx content was a little higher. The enzyme was inhibited by heavy metals, such as Hg²⁺, Cu²⁺, and Ag⁺, as well as pCMB; the activity was restored by incubating with mercaptoethanol. Fructose and amines including Tris and aniline had inhibitory effects.

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The growth of a newly isolated strain of Pediococcus sp., designated ISK-1, was very slow and the concentration of cells in the medium remained low. Fermentation with an initial 30 g/liter glucose required about 60h. To stimulate fermentation, we attempted to optimize the medium by flask culture and jar fermentation tests. Mevalonic acid and mieki (soy bean hydrolyzate) stimulated fermentation and increased the rate of formation of DL-lactate. Kinetic analysis of the fermentation showed that mevalonic acid markedly increased the specitic glucose consumption rate and the specific lactate production rate. Mieki and mevalonic acid had a synergistic effect, but the effect of mevalonic acid was different from that of mieki.

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The biological activities of 7 microbial extracellular glycolipids including mannosylerythritol lipid (MEL)-A, MEL-B, polyol lipid (PL), rhamnolipid (RL), sophorose lipid (SL), succinoyl trehalose lipid (STL)-1, and STL-3 were investigated. All glycolipids except for RL were found to induce cell differentiation instead of cell proliferation in the human promyelocytic leukemia cell line HL60. To identify the differentiation direction of the induced cells, the leukocyte esterase activities were cytologically investigated, and the results showed that MEL-A, MEL-B, and PL induced HL60 to differentiate into granulocytes, while SL, STL-1, and STL-3 induced differentiation into monocytes. The 6 effective glycolipids also increased nitroblue tetrazolium (NBT) reducing ability, which is a common differentiation-associated characteristic in monocytes and granulocytes. Furthermore, it was also observed that these 6 glycolipids inhibited the activity of phospholipid- and Ca²⁺ -dependent protein kinase. Additionally, the 6 effective glycolipids also induced the human myelogenous leukemia cell line K562 and the human basophilic leukemia cell line KU812 to differentiate into monocytes, granulocytes, and megakaryocytes.

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Endo-1, 4-β-D-xylanase (EC 3.2.1.8) was purified from viscera of a fresh-water mollusc, Pomacea insularus (de Ordigny). The purified enzyme, with a molecular weight of 47, 000, gave a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amino-terminal sequence was Ala-Ala-Gly-Ala-Gly-Val-Thr-Ser-Glu-Lys-Asp-Arg-Leu-Arg-Arg-Ser-Asp-Lys-Thr-Val-His-Val-Asn-. The enzyme was stable from pH about 4.5 to 9.5 and had its maximum activity at pH about 5.5. The puritied enzyme produced X2, X3, X4, and larger xylooligosaccharides from birchwood xylan. The enzyme activity was greatly inhibited by Ag⁺, Hg²⁺, Cu²⁺, N-bromosuccinimide, and p-chloromercuri-benzoic acid, On the other hand, the enzyme activity was greatly elevated by the addition of chloride ion.

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Adenosine 5'-phosphosulfate (APS) sulfotransferase is thought to be an enzyme that transfers the sulfo-group of APS to a carrier compound with a thiol group in the assimilatory sulfate reduction pathway of higher plants. We developed a rapid, non-radioactive assay for APS sulfotransferase. Sulfite released by APS sulfotfansferase reaction in the presence of excess dithiothreitol was further converted to cysteine by coupling with yeast sulfite reductase and cabbage O-acetylserine(thiol)lyase. The cysteine thus formed was measured colorimetrically. By this method, 5 to 300 nmol of sulfite could be assessed. When the method was applied to APS sulfotransferase, the enzyme activity was APS-dependent with the partially purified enzyme. We could also detect APS sulfotransferase activity in some higher plants by this method.

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Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and phosphoenolpyruvate kinase (EC 4.1.1.49<ATP>, PEPK) were detected in cells of Rhodopseudomonas sp. No. 7 grown photoanaerobically on ethanol and acetate. The activity of PEPC was about 3 times higher in the ethanol-grown cells than the acetate-grown cells. PEPC was purified as an electrophoretically homogeneous and stable protein (Mr, about 400, 000; subunit Mr, about 102, 000). The enzyme absolutely required Mn²⁺ or Mg²⁺ for the appearance of its activity. Acetyl CoA (40 μM) reduced the K_m against phosphoenolpyruvate and HCO^-₃ to about 1/20 (0.23mM) and 1/2 (1.7mM), respectively. At that time, the V_max increased to about 30 times (90 μmol/min/mg protein). In the presence of acetyl CoA the circular dichroism spectrum of the enzyme showed the decrease of an α-helix structure. The enzyme was not activated by fructose-1, 6-bisphosphate. The enzyme was inhibited by aspartate (K_i, 0.208mM). Besides, the enzyme was inhibited by nucleotides (ATP and GTP). The enzyme activity was activated to about 15 times by 3.25M ethanol.

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Yeast Saccharomyces cerevisiae cells were cultured synchronously and the change of platelet-activating factor (PAF) production during the cell cycle was investigated at each phase of the cycle. The basal PAF contents of diploid AKU4103 cells in G1 and M phases were higher than those of cells in S phase. Both diploid and haploid strains showed the same level of PAF production in response to the calcium ionophore A23187. A23187-stimulated PAF productions of cells in G1 and M phases were about 20 times higher than that of cells in S phase. The contents of PAF precursor in G1 and M phases cells of AKU4103 were higher than those in S phase cells, and the ratio of A23187-stimulated PAF to the precursor was highest in G1 phase cells. We also examined the change in a PAF-synthesizing enzyme, acetyltransferase, activity during the cell cycle using a microsomal fraction. Specific activity was the highest at G1 phase, and total activity was higher at M phase. The enzyme activities of cells in S phase of strains AKU4103 and RAY-3Aa were one-third and one-tenth of those in G1 phase of corresponding cells, respectively. These results suggest that PAF production was higher at G1 and M phases and lower at S phase, and changes in PAF productivity during cell cycle were related to the precursor contents and the synthesizing enzyme activities in those cells. These data suggest that PAF may control the cell cycle phase in budding yeast.

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An extracellular endo poly(β-D-1, 4-mannuronide) lyase of Dendryphiella salina IF 32139 was purified to homogeneity by Q Sepharose FF and Sephacryl S-200 HR column chromatographies. The purified enzyme had a molecular weight of 35, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.65 by isoelectric focusing. The optimum pH and temperature for enzyme activity were pH 5.0 and 45°C, respectively. The enzyme was stable from pH 4 to 10 and at temperature below 40°C. Some divalent cations, Ca²⁺, Mn²⁺, and Zn²⁺, increased the enzyme activity. Hg²⁺ and NBS strongly inhibited the activity. This enzyme susceptibly degraded poly-M, produced a wide range of 4, 5-unsaturated oligomannuronic acids, and further degraded these unsaturated oligomannuronic acids to produce the unsaturated monomer and dimer as final products.

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The isolated N-lobe (1-332) and C-lobe (342-686) of ovotransferrin were each found to form an opaque gel by incubation with 70 mM 2-mercaptoethanol at 37°C for 24 h, suggesting that a protein conformational change due to extensive disulfide reduction was the initial mechanism prior to subsequent intermolecular protein aggregation forming gel networks. To investigate the conformational state of an extensively disultide-reduced protein, the structural characteristics of a two-disulfide protein form that had been shown to be produced by extensive disulfide reduction of the N-lobe [H. Yamashita, T. Nakatsuka, and M. Hirose, J Biol. Chem., 270, 29806-29812 (1995)] were analyzed, as a model protein, by the protein fragmentation approach. When the two-disulfide form was proteolyzed with chymotrypsin, a protease-resistant 8-kDa fragment was produced. This fragment was purified by ion-exchange column chromatography, and chemical analyses and matrix-assisted laser desorption ionization time-of-flight mass spectrometry clearly showed that the 8-kDa fragment comprised Ala1-Tyr72 of ovotransferrin. The fragment was found by amino acid analysis to retain the two intra-chain disulfide bonds intact. Far-UV CD spectrum analyses revealed that the 8-kDa fragment retained a native-like folded conformation. These data are consistent with the view that the N-terminal degment, Ala1-Tyr72, assumed a local native-like conformation in the two-disulfide form of the ovotransferrin N-lobe.

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Four indophenol-reducing compounds, USF-406A, B, C, and D, were isolated from a culture filtrate of the fungus, Mortierella sp. USF-406 strain by chemical screening. Their structures were elucidated by spectroscopic evidence and chemical degradation. Two were new phenol compounds, N-(4, 6-dihydroxy-2, 3, 5-trimethylbenzoyl)-glycine and its methyl ester, and the others were their known derivatives. Biosynthetic studies, using ¹³C-labelled compounds and ¹³C-NMR spectroscopy, showed they were derived from the same tetraketide precursor. Their antioxidative activities were measured, and they acted as electron donors and also as radical scavengers. The production of these phenol compounds with antioxidative activity might have be related to the production of unsaturated fatty acids by the same fungus.

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Dihydroflavonol taxifolin and its glycoside, astilbin, from Engelhardtia chrysolepis inhibited rat lens and recombinant human aldose reductase. Taxifolin also inhibited sorbitol accumulation in human red blood cells. Furthermore, this dihydrotlavonol aglycone maintained the clarity of rat lens incubated with a high concentration of glucose. These dihydroflavonols may be effective for preventing osmotic stress in hyperglycemia.

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Endopolygalacturonase (endoPG) I was obtained from Stereum purpureum by an improved easier purification procedure. It was found that EndoPG I consisted of three glycosilated proteins with the same isoelectric point and different molecular masses, 42, 45, and 48kDa, respectively. However, the enzymatic deglycosilation product of endoPG I gave a single band at the position corresponding to 39kDa on SDS-PAGE. Furthermore, the N-terminal amino acid sequences of three endoPGs were identical one another up to 20 residues. A cDNA library was constructed and positive cDNA clones encoding endoPG I were isolated by using antibody raised against the purified endoPG I. Nucleotide sequence analysis of the cDNA disclosed a 1212-bp open reading frame that encoded 403 amino acid residues. The N-terminal amino acid sequence (residues 1-20) of endoPG I coincided with the deduced amino acid sequence starting from the 25th residue. Therefore, the sequence of the first 24 residues represented a signal peptide and the remaining sequence, consisting of 379 residues, was the mature protein with molecular mass of 39.1 kDa.The deduced sequence of endoPG I showed 30-45% similarity in comparison with those of bacterial and fungal endoPGs, and the sequence of putative active site residues reported for the endoPGs was highly conserved in the sequence of endoPG I.

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(+)-Phrymarolin I was stereoselectively synthesized from (R)-(+)-3-hydroxybutanolide that had been prepared from (+)-malic acid. The procedure is more efficient than our previous synthesis in terms of fewer reaction steps and the easier availability of the starting material.

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Using a calorimeter with 24 sample units the heat evolved during incubation of yeast cultures at 30°C was detected in the form of growth thermograms. Ethanol and methanol added to the culture medium produced changes in the growth thermograms that could be analyzed to calculate the 50% inhibitory concentration (K_i) and minimum inhibitory concentration (MIC). Correlation of the heat evolution curves with the number of cells and the turbidity of the culture was found to be very good. It was found that addition of ethanol and methanol up to 7.65% had clear effects of inhibition on growth of all yeast strains studied, reducing the growth rate constant and delaying growth. However, the amounts of ethanol produced from the nutrients available in the culture vial was only little affected by the initial addition of up to 7.65% (v/v) of ethanol or methanol in the medium.

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Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and an EPO-fixed gel for sEPOR. The atlinity purification efficiently removed unwanted proteins, giving samples with suffciently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.

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Mucus glycoprotein (RGP) was purified and characterized from the skin mucus of rainbow trout, Salmo gairdneri. RGP was found to contain 30.1% NeuAc, 26.0% GalNAc, 5.0% Gal, and 26.0% amino acids. The protein moiety of RGP is very rich in Thr (32.4 mol%). Neither NeuGc nor KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) was found in RGP. Alkaline borohydride treatment of RGP yielded a major disaccharide alditol, NeuAcx2→6GalNAc-ol and more than 4 minor oligosaccharide alditols including NeuAc→(GalNAcx1→)GalNAc-ol. It was evident that an average RGP molecule has approximately 500 NeuAc-containing oligosaccharide chains, which are attached to the Thr and Ser residues of the protein moiety and spaced at an average of 3 amino acids apart.

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Enterobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Enterobacter sp. G-1. To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60, 000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10 mM EDTA, but was not inhibited by Ca²⁺ (<50 mM) or NaCl (<400 mM). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.

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The complete amino acid sequence of pokeweed lectin-B (PL-B) has been analyzed by first sequencing seven lysylendopeptidase peptides derived from the reduced and S-pyridylethylated PL-B and then connecting them by analyzing the arginylendopeptidase peptides from the reduced and S-carboxymethylated PL-B. PL-B consists of 295 amino acid residues and two oligosaccharides linked to Asn96 and Asn139, and has a molecular mass of 34, 493 Da. PL-B is composed of seven repetitive chitin-binding domains having 48-79%) sequence homology with each other. Twelve amino acid residues including eight cysteine residues in these domains are absolutely conserved in all other chitin-binding domains of plant lectins and class I chitinases. Also, it was strongly suggested that the extremely high hemagglutinating and mitogenic activities of PL-B may be ascribed to its seven-domain structure.

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Glycosyl-trehaloses with an isomaltosyl residue were synthesized by α-glucosidase from Aspergillus niger by using maltotetraose as a glucosyl donor and trehalose as the acceptor. The one trisaccharide and two tetrasaccharides formed were isolated by successive column chromatography. The results of an enzymatic digestion, methylation analysis, and ¹³C-NMR studies indicated that these oligosaccharides were α-isomaltosyl α-glucoside, α-isomaltotriosyl α-glucoside and α-isomaltosyl α-isomaltoside. These oligosaccharides were not fermented to an acid by Streptococcus mutans, and they effectively inhibited water-insoluble glucan synthesis from sucrose by glucosyltransferase. In an in vitro utilization test with human intestinal bacteria, these oligosaccharides were predominantly utilized by Bifidobacteria.

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The Saccharomyces cerevisiae iki mutants show an insensitive phenotype to the pGKL killer toxin, and we have cloned some IKI genes by complementation of this phenotype [Kishida et al., Biosci. Biotech. Biochem., 60, 798-801 (1996)]. Here, we identified and characterized the IK and IKI3 genes. DNA sequencing of the genes showed that both have 100% identity with hypothetical genes identified by the yeast genome project, YHR187w (481, 911-480, 985 in chromosome VIII) for IKI1, and YLR384c (888, 852-892, 898 in chromosome XII) for IK. Both are novel genes with no significant identity with other known genes and they do not belong to any homology domain group, gene family, or superfamily. The disruption of IKI1 is not lethal, but growth of the disruptant was slower than that of the wild type at all temperatures examined. The disruptant was the killer-insensitive phenotype. The sequence of the IKI1 gene predicted a hydrophilic protein with a molecular mass of 35 kDa (309 amino acids). A 35-kDa protein band was also detected by immunoblotting the 25, 000 × g pellet fraction of the wild type yeast cell lysate. Disruption of the IKI3 gene is also non-lethal and it has the killer-insensitive phenotype. Iki3p may contain a transmembrane domain near the NH₂-terminal region (97-113 residues in a total of 1349 amino acids).

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We have discovered a novel metalloproteinase, which has high activity at low temperatures, from the culture supernatant of a marine bacterium. The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase, named almelysin, was purified to homogeneity from the cultured supernatant at 10°C by two column chromatographies. About 20mg of purified almelysin was obtained from 18.4 liters of the culture supernatant. The molecular mass of almelysin was estimated to be 28 kDa by SDS-PAGE and the isoelectric point was 4.3. The optimum pH for activity of almelysin was pH 8.5-9.0 and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N³-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)[A₂pr(Dnp)]-Ala-Arg-NH₂ as substrates, respectively. Almelysin was stable between pH 7.5-8.0 and below 40°C. The optimum temperature for the activity was observed to be 40°C using both casein and MOCAc-Pro-Leu-Gly-Leu-A₂pr(Dnp)-Ala-Arg-NH₂ as substrates. The activity of almelysin was inhibited by such metallo chelators as EDTA and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved the Ala¹⁴-Leu¹⁵ bond and Phe²⁴-Phe²⁵ bond, and secondarily the Tyr¹⁶-Leu¹⁷ bond in oxidized insulin B-chain. However, almelysin could not cleave the His⁵-Leu⁶, His¹⁰-Leu¹¹, and Gly²³-Phe²⁴ bonds, which were cleaved by other metalloproteinases. These results indicate that the substrate specificity of almelysin is different from other metalloproteinases. Interestingly, Alteromonas sp. No. 3696 strain produced another proteinase as well as almelysin at 25°C.

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Sodium linoleate (LA) and sodium docosahexaenoate (DHA) were oxidized in an aqueous phase with or without Tween 20. The oxidative stability of LA with Tween 20 was slightly less than that of LA without Tween 20. In contrast, when DHA was dispersed in a buffer containing Tween 20, the stability of DHA was markedly increased, suggesting a specific effect of Tween 20 on protecting DHA against oxidation in aqueous micelles.

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Functional role of Asn219 of aqualysin I, a thermostable serine protease from Thermus aquaticus, was investigated by using site-directed mutagenesis. Replacement of Asn219 with serine increased the catalytic efficiency (k_cat/K_m) for synthetic peptide substrates about twice as much as that of the wild type, while threonine replacement caused a slight decrease in the efficiency. Such replacements resulted in a significant change of k_cat rather than K_m, indicriting that the side chain in the vicinity of the catalytic residue Ser222 affects the catalytic rate constant.

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We have already identified N-(β-D-glucopyranosyl)nicotinic acid, which was isolated from cultured tobacco cells, as a novel niacin metabolite. The enzyme activity for the biosynthesis of this compound, UDP-glucose: nicotinic acid-N-glucosyltransferase, was found in cell-free extracts from cultured cells of tobacco (Nicotiana tabacum XD-6) and properties were investigated in this experiment. The general catalytic properties were as follows: optimum temperature was 25°C, optimum pH was 6.1, the K_m for NiA and UDP-glucose were calculated at 220 μM and 4.3 mM, respectively. The enzyme activity was lowered by heavy metal ions, isonicotinic acid, benzoic acid, and 6-hydroxynicotinic acid. The roles of niacin metabolites in cultured tobacco cells are discussed in relation to the enzyme.

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The effects of food additives on immunoglobulin produced in rat splenic lymphocytes were examined. The xanthene dye, Rose Bengal, enbanced IgE production, while inhibiting the production of IgG and IgM, at 50μM. Among the xanthene dyes, Rose Bengal having 4 iodine and 4 chlorine atoms exerted the highest Ig production-regulating activity in splenocytes, and dihalogenated fluorescein, a diiodo compound, exerted similar activity, while the dichloro and dibromo compounds did not. These results suggest that halogen atoms, especially the iodine atom, in xanthene dyes play an important role in regulation of Ig production.

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A glass microfiber filter was found useful for detection of the complex of lysyl-tRNA synthetase from Bacillus stearothermophilus with lysyladenylate. Detected c.p.m. values of the ³H-labeled complex trapped by the glass microfiber filter was about 2-fold higher than those by nitrocellulose filter and about 3-fold higher than those by DEAE-cellulose filter because of its higher counting efficiency. The use of the glass fiber filter is more suitable for the lysyl-tRNA synthetase assay than the use of either nitrocellulose or DEAE-cellulose filters, which were used for other aminoacyl-tRNA synthetases. Effects of pH on the filter assay of lysyl-tRNA synthetase activity using the glass microfiber filter were also examined.

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Zinc (Zn)-resistant Alethylobacterium strains were isolated from soils collected in Japan. Although the soils used had never been polluted with Zn, Zn-resistant Methylobacterium-like red colonies were detected at 10² to 10³ per gram of dry soil. The 16S ribosomal DNA sequence of the representative eleven strains were most similar to Methylobacterium radiotolerans. Furthermore, all of Methylobacterium strains from culture collections showed moderate or high Zn resistance. Thus, Zn resistance might be an inherent characteristic in Metethylobacterium species.

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A cysteine proteinase was purified from dormant seeds of wheat (Triticum aestivum, cultivar Norin 61) and its molecular mass was estimated to be about 23kDa by gel filtration. The K_m of this proteinase at pH 5.5 was calculated as 2 μM for Z-Phe-Arg-MCA, and its activity was inhibited by cysteine proteinase inhibitors including antipain, E-64, and leupeptin. Oryzacystatin-I, a proteinaceous cysteine proteinase inhibitor in rice seeds, also inhibited its activity.

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For potential application in the economic production of bacterial cellulose, cellulose-producing Acetobacter strains capable of using sucrose as a carbon source were screened for using corn steep liquor as a nitrogen source. A total of 1500 strains were isolated and four were selected through static and shaking flask cultures. Much higher cellulose accumulation was observed in a jar fermentor using these strains in sucrose-CSL medium than seen using the previously isolated strain, BPR2001, in fructose-CSL medium. It was found that these strains also had high cellulose production from glucose. The pH of the cultures of the isolated strains did not decrease, and it was suggested that these strains have low glucose-oxidiring ability.

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The cytoagglutinating activity of abrin-b, a toxic lectin isolated from Abrus precatorius seeds, against cultured cell strains derived from accute lymphoblast leukemia (ALL) was investigated by visible (VIS) spectroscopy. Upon addition of abrin-b, the turbidity at 600 nm of cell suspension decreased and this change could be recorded as the cytoagglutination curve. From this curve, the cytoag-glutination velocity (CV) and cytoagglutination intensity (CI) of each cell strain was measured. Each cell strain showed the respective CV and CI values and the cell strains derived from the T cell line were strongly agglutinated by abrin-b compared with those derived from the B cell line. Further, it has become apparent that the cytoagglutinating activity increased with an increase in the order of the differentiation of cell strains.

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Seven coumaric compounds analogous to aurapten were synthesized. Their spasmolytic activity against Ba²⁺, acetylcholine and histamine was evaluated to investigate their structur-activity relationship. The results of the bioassay demonstrated the important roles of the cis type of double bond at C-2' and the epoxide between C-6' and 7'.

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(+)-Paulownin, a furofuran lignan from Paulownia tomentosa, was stereoselectively synthesized from (R)-(+)-3-hydroxybutanolide in 12 steps with a yield of 4.4%.

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α-Clausenan [3-methyl-2-(3-methyl-1, 3-butadienyl)furan], which is found in Clausena willdenovii, was synthesized by starting from methyl 3-(3-methyl-2-furanyl)propionate in a 34% overall yield in 4 steps.

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This paper describes with the multiformity of intracellular products among the recombinants obtained by interspecific protoplast fusion between Streptomyces griseus and S. griseoruber and between S. griseus and S. rochei. The products were extracted from the mycelium with methanol and analyzed by thin-layer chromatography using three detection methods. Detection with anisaldehyde-sulfuric acid reagent and Ehrlich's reagent showed that 29-37% of the recombinants produced new products different from those of the parental species, while about 50% of the recombinants did not produce any detectable substances. In a bioautogram against Bacillus subtilis, about 50% of the recombinants showed clear antibiotic activity in their methanol extracts, while the parental species did not. The distribution of the products (parental, mixed, new, or not expressed types) among the recombinants is discussed compared with that of taxonomic allelo-characters.

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An asymmetric total synthesis of (+)-coronafacic acid, starting from (R)-(+)-4-acetoxy-2-cyclopen-1-one as a chiral source, was accomplished. Construction of the 1-hydrindanone framework was carried out by using intramolecular 1, 6-conjugate addition as the key step. Coupling between (+)-coronafacic acid and protected coronamic acid, and subsequent deprotection provided (+)-coronatine. This is the first asymmetric total synthesis of (+)-coronatine.

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To study the role of osteopontin, we did cell adhesion and ALP assays of rat bone marrow osteoblastic cells (RBMO) on collagen Type I and osteopontin surfaces. The RBMO proved to adhere much more strongly to the osteopontin and to have higher ALP activity on the osteopontin, which suggests that pre-osteoblasts differentiate into osteoblasts that form bone by recognizing osteopontins.

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