Scab disease significantly damages potato and other root crops. Streptomyces scabiei, S. acidiscabiei, and S. turgidiscabiei are the best-known causal agents of this disease. We have developed a novel genotyping method for these potato scab pathogens using multiplex PCR, whose benefits include rapid and easy detection of multiple species. We designed a species-specific primer set (6 primers, 3 pairs) for the 16S rRNA genes and 16S–23S ITS regions of these potato scab pathogens. The specificity of the primer set was confirmed by testing 18 strains containing potato scab pathogens, other Streptomyces species, and strains of other genera. The application of the developed method to potato field soil and potato tissue samples resulted in the clear detection and identification of pathogens. Since this method is applicable to a large number of environmental samples, it is expected to be useful for a high-throughput analysis of soil and plant tissues of scab disease.

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All stereoisomers of goniodiol were synthesized from yeast-reduction products. The C-6 chiral centers were converted from the chiral centers of the yeast-reduction products. Stereoselective conversion of the alkene, which had been prepared from the yeast-reduction product, to glycol constructed the C-7 and C-8 stereochemistry. (+)-Goniodiol and 7-epi-(+)-goniodiol showed the highest antibacterial activity (MIC, 3.1 mM) against Yersinia intermedia.

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We identified a new radical scavenger, ansaetherone (C₂₆H₃₃NO₇), from a culture of the Streptomyces sp. USF-4727 strain. In our previous study, it was shown that this strain produced four lipoxygenase inhibitors, tetrapetalones A, B, C and D. The chemical structure of ansaetherone was elucidated by the spectroscopic method, indicating that this compound was constructed with an aglycon and a sugar moiety. This chemical structure suggested that ansaetherone was related to the tetrapetalones. This finding provided information regarding tetrapetalone biosynthesis. Ansaetherone showed radical scavenging activity with an ED₅₀ value of 300 μM in our assay.

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Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.

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(RS)-β-Ionol and (RS)-2-methyl-4-octanol were resolved by using (S)-2-methoxy-2-(1-naphthyl)propanoic acid [(S)-MαNP acid]. The specific stereochemistry of each MαNP ester was elucidated by 2D NMR analyses, and shielding by the 1-naphthyl group was observed in both the ¹H- and ¹³C-NMR spectra. Solvolysis of the individual (S)-MαNP esters gave four single-enantiomer alcohols. The normal-phase HPLC elution order of each MαNP ester is also discussed.

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Two new flavonol glycosides and three known flavonoids were isolated from seeds of Camellia semiserrata Chi. The structures of these new flavonol glycosides were established as kaempferol 3-O-[(2'''',3'''',4''''-triacetyl)-α-L-rhamnopyranosyl(1→3)(2''',4'''-diacetyl)-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside] and kaempferol 3-O-[(3'''',4''''-diacetyl)-α-L-rhamnopyranosyl(1→3)(2''',4'''-diacetyl)-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside] by spectroscopic methods. The estrogenic activity of these compounds was investigated by a recombinant yeast screening assay.

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In the course of our screening for a new anti-tumor substance, the bisabolane sesquiterpenoid endoperoxide, 3,6-epidioxy-1,10-bisaboladiene (EDBD), was isolated from the edible wild-plant, Cacalia delphiniifolia. EDBD showed cytotoxicity toward human chronic myelogenous leukemia K562 and human prostate carcinoma LNCaP cell lines with IC₅₀ values of 9.1 μM and 23.4 μM, respectively. DNA fragmentation and condensation of chromatin, the hallmarks of apoptosis, appeared in K562 cells after an 18-h treatment with EDBD. α-Curcumene, a bisabolane sesquiterpene that lacks the endoperoxide moiety of EDBD, also showed cytotoxicity toward both K562 and LNCaP cell lines at over a 10-times higher dose than that of EDBD. The results indicate the importance of the endoperoxide structure within EDBD to its anti-tumor activity in vitro.

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Abnormal polyglutamine (polyQ) expansion in the N-terminal domain of the human androgen receptor (hAR) is known to cause spinobulbar muscular atrophy (SBMA), a hereditary human neurodegenerative disorder. To explore the molecular mechanisms of neurodegeneration in SBMA, we genetically screened modulators of neurodegeneration in a Drosophila SBMA experimental model system. We identified hoip as an accelerator of polyQ-induced neurodegeneration. We found that hoip forms a complex with 18s rRNA together nop56 and nop5 proteins, whose human homologs are known to form a snoRNP complex involved in ribosomal RNA processing. Significantly, the levels of mutant polyQ-hAR were up-regulated in a mutant line overexpressing hoip. Consistently, severe neurodegeneration phenotype (rough eye) was also observed in both nop56 and nop5 overexpression mutant lines. These findings suggest that the process of neurodegeneration induced by abnormal polyQ expansion in the hAR may be regulated by the activity of snoRNP complex.

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Glutamate decarboxylase (GAD) is an enzyme that synthesizes γ-aminobutyrate (GABA), a major inhibitory neurotransmitter in the central nervous system. Post-translational modification of GAD, such as N-terminal blockage, phosphorylation-dephosphorylation, and palmitoylation, is an important factor in the biological activity of GAD. In order to address the significance of post-translational events on GAD, we thought it crucial to obtain a non-recombinant form of GAD. In this study, we attempted to isolate GAD protein from the monkey brain, a model animal close to the human that has not been studied. Monkey brain was homogenized, fractionated with ammonium sulphate, and applied to a series of chromatographic steps, including hydrophobic, ion-exchange, and gel filtration. Purified GAD showed a single band on SDS–PAGE, and the enzyme was found to have a molecular weight of 61,000 and exhibited 1,100 nmol/min/mg of specific activity. It had an optimal pH of 7 and optimal thermal stability at 40 °C.

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Granzyme A (GzmA) is a serine protease (trypsin-like specificity) produced in cytotoxic lymphocytes. This enzyme is believed to enter virus-infected cells and growing tumors and induce apoptosis, but the roles of GzmA expressed in lymphocytes scattered through the epithelial layer of the normal small intestine are unknown. In the present study, recombinant rat GzmA (rGzmA) was found to cause morphological changes and detachment of a non-transformed rat small-intestinal epithelial cell line IEC-6, although the rGzmA-treated cells detached as aggregates with no changes characteristic of apoptosis. rGzmA-induced deformation and detachment occurred in IEC-6 cells plated with collagen type IV and fibronectin, but not in those plated with laminin. These findings suggest that GzmA in the normal small intestine participates in the reduction of adhesion between epithelial cells and basement membranes, through its ability to cleave extracellular matrix components.

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The recombinant two-kringle domain of human tissue-type plasminogen activator (TK1-2) was found to inhibit angiogenesis and tumor growth. Recently, we found that TK1-2 inhibits adhesive differentiation of endothelial progenitor cells, and its contribution to tumor angiogenesis. In this study, we investigated the effects of TK1-2 on extracellular matrix-induced adhesion, signaling, and migration in order to understand the mechanism of action of TK1-2. When human umbilical vein endothelial cells were pretreated with TK1-2 and then allowed to adhere to immobilized fibronectin, vitronectin, or gelatin, cell adhesion to all the tested matrices decreased dose-dependently upon TK1-2 treatment. TK1-2 also inhibited the formation of actin stress fibers and focal adhesions upon attachment to each matrix. Moreover, fibronectin- and vitronectin-induced endothelial cell migration was dose-dependently inhibited by TK1-2. TK1-2 also suppressed fibronectin-induced ERK1/2 phosphorylation. Hence the results suggest that disturbance of extracellular matrix-induced adhesion, signaling, and migration of endothelial cells is involved in the anti-angiogenic activity of TK1-2.

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Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent promising candidates for the development of future cellular therapy strategies. MSCs have been found to be able to differentiate into various tissues. One of the primary limitations in our understanding of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on proteins that were differentially expressed during osteogenic differentiation of hUCB-MSCs. And we analyzed the protein expression inherent to osteogenic differentiation by two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed spots were observed between the two groups (before and after differentiation) on the 2-DE map. These might also be proved as useful cytosolic biomarker proteins for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.

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Human cystathionine β-synthase (CBS) catalyzes a pyridoxal 5′-phosphate (PLP) dependent β-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 Å away from heme. A possible explanation of our results is discussed on the basis of CBS structure.

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The tphA1_II and tphA2_IIA3_II genes of Comamonas sp. E6 perhaps code for the terephthalate (TPA) 1,2-dioxygenase (TPADO). To characterize E6 TPADO, these genes were expressed in a His-tagged form in Escherichia coli, and the recombinant proteins were purified. TPADO activity was reconstituted from TphA1_II and TphA2_IIA3_II, indicating that TPADO consists of a reductase (TphA1_II) and a terminal oxygenase component (TphA2_II and TphA3_II). TphA1_II contains FAD, and the presence of a plant-type [2Fe-2S] cluster was suggested. These results indicate that TPADO is a class IB aromatic ring-hydroxylating dioxygenase. NADH and NADPH were effective as electron donors for TphA1_II, but NADPH appeared to be the physiological electron donor, based on the kinetic parameters. TPADO showed activity only toward TPA, and Fe²⁺ was required for it. The K_m values for TPA and the V_max were determined to be 72±6 μM and 9.87±0.06 U/mg respectively.

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cDNA encoding a novel defensin (BmDefensinB) was cloned from the fat body of the silkworm, Bombyx mori, and gene expression was analyzed. BmDefensinB showed typical structural characteristics of invertebrate defensins. Phylogenetic and bootstrap analyses indicated that it has no orthologs, whereas previously reported BmDefensinA is the ortholog of Spodoptera frugiperda (Sf)Spodoptericin. The BmDefensinB gene was expressed tissue-specifically in the fat body and was strongly activated by bacteria such as Escherichia coli and Bacillus subtilis, and by an entomopathogenic fungus Beauveria bassiana. In contrast, the BmDefensinA gene was expressed to a much lesser extent. Expression of the BmDefensinB gene was strongly stimulated by B. mori Rel proteins RelB and Relish, supporting the observation that this gene is activated by E. coli, B. subtilis, and B. bassiana. These results suggest that BmDefensinB gene expression is controlled through both the Toll and the Imd pathway, and that this gene plays an important role in B. mori immune reactions against infection by bacteria and fungi.

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Atomic force microscopy (AFM) observation of a crystal surface of the thermostable isocitrate dehydrogenase (ICDH) from a thermophilic eubacterium, Thermus thermophilus HB8, suggested that the crystal consists of huge homo-complexed ellipsoidal bodies of the protein, with averaged long- and short-axis diameters of 18.6 nm and 10.9 nm respectively. Thick diamond-shaped crystals of about 0.4 mm on the longest axis were obtained by the vapor diffusion method from a solution of 100 mM sodium cacodylate, pH 6.6–8.4, containing 1.4 M sodium acetate as the precipitate, and diffracted X-rays at 3.7 Å resolution. The crystals belonged to the monoclinic lattice type with space group C2 and had cell dimensions of a=495.5, b=189.2, c=336.2 Å, and β=126.4°, indicating that an asymmetric unit contained more than 33 molecules with a molecular mass of 54.2 kDa. Calculations based on data obtained by the X-ray method showed good agreement with AFM observation. These results suggest the possibility that the residing T. thermophilus HB8 ICDH molecules are piled one on top another as a preformed supramolecular nano-architecture in the crystal lattice. The system appears suitable for further investigation using a bottom-up approach to the self-associated construction of nano-architectures with proteins.

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This study compared the antinociceptive and anti-inflammatory effects of various parts of Broussonetia papyrifera (L.) L’Herit. ex Vent. (BP, Moraceae) by chemical-induced pain and inflammation in rodents. All BP parts (1 and 2 g/kg, p.o.) effectively inhibited writhing responses induced by 1% acetic acid. The BP radix, leaf, and fruit effectively inhibited the late-phase licking responses caused by 1% formalin. But only the BP radix and fruit reduced the edema induced by 1% carrageenan at 1–2 h. Furthermore, the BP radix reduced the abdominal Evan’s blue extravasations caused by inflammatory mediators, including serotonin and sodium nitroprusside. Finally, the radix had the highest contents of betulin and betulinic acid among all BP parts. In conclusion, the radix is the better medicinal BP part possessing antinociceptive and antiinflammatory effects, and its anti-inflammatory effects are partially related to the inhibition of vascular permeability via autocrines and nitric oxide.

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The mercury resistance module of Bacillus transposon TnMERI1 is regulated by three operator/promoter regions (O/P_merB3, O/P_merR1, and O/P_merR2) and two regulatory proteins (MerR1 and MerR2) encoded by the module itself. To clarify the roles of MerR1 and MerR2 in the regulatory mechanism, both proteins were overexpressed and purified. MerR1 bound the regulatory regions O/P_merB3 and O/P_merR1, with a preference for O/P_merB3 as measured on in vitro gel shift assays. However, MerR2 bound O/P_merR2, as revealed by gel shift and restriction endonuclease protection assays. The transcriptional start sites of O/P_merB3 and O/P_merR2 were determined by rapid amplification of 5′-cDNA ends (5′-RACE) in the TnMERI1 original host, Bacillus megaterium strain MB1. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays showed that O/P_merB3 and O/P_merR1 were induced in the presence of Hg²⁺ but not O/P_merR2. It was concluded that MerR1 regulates O/P_merB3 and O/P_merR1, while MerR2 regulates O/P_merR2.

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A methioninase inhibitor from Myrsine seguinii was purified and identified as myrsinoic acid B. Its inhibitory activities as to crude methioninase from periodontal bacteria such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Treponema denticola were determined. The IC₅₀ values were 10.5, 82.4, and 30.3 μM respectively.

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Cellobiose phosphorylase from Clostridium thermocellum catalyzed the β-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl β-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl β-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.

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We investigated changes in the sub-cellular distribution of glycelaldehyde-3-phosphate dehydrogenase (GAPDH) after X-ray irradiation in HeLa cells. Twenty-four h after irradiation at 5 Gy, nuclear GAPDH levels increased 2.6-fold, whereas total GAPDH levels increased only 1.2-fold. Knockdown of GAPDH using specific small interfering RNA (siRNA) led to sensitization to X-ray-induced cell death. These results suggest that GAPDH plays a role in the radioresponse.

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Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.

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Lactate dehydrogenase A4 (LDH-A4) was purified for yak skeletal muscle. Michaelis constant (Km) analysis showed that yak LDH-A4 for pyruvate was significantly higher than that of cattle. cDNA cloning of LDH-A revealed two amino acid substitutions between yak and cattle. We suggest that the higher Km of yak LDH-A4 might be a result of molecular adaptation to a hypoxic environment.

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The L-tryptophan decarboxylase (TDC) gene of rice was heterologously expressed in various organisms. Transgenic rice overexpressing TDC showed accumulation of serotonin upon 5-hydroxytryptophan treatment, which was consistent with the in vitro 5-hydroxytryptophan decarboxylase enzyme activity of purified recombinant rice TDC in a pyridoxal phosphate-dependent manner. Recombinant yeast harboring TDC produced serotonin at the expense of the endogenous 5-hydroxytryptophan levels.

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An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158–163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 °C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 °C of HPH to 53 and 58.8 °C respectively. Specific activities and steady-state kinetics measured at 25 °C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.

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Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.

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Granzyme A (GrA) is a serine protease produced in cytotoxic lymphocytes, lung epithelial cells (alveolar type-II cells), and alveolar macrophages. In the present study, recombinant rat GrA (rGrA) was found to cause rounding and detachment of an alveolar type-II epithelial cell line, A549. Also, rGrA stimulated release of a neutrophil chemoattractant, interleukin-8, from the cells, via a mechanism involving microtubule disruption, probably resulting from reduction of cell adhesion to culture dishes. These findings suggest that GrA might be involved in the pathogenesis of certain lung diseases characterized by loss of alveolar wall structures, neutrophil accumulation, and chronic inflammation.

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In the biosynthesis of Fusarium trichothecenes, the C-3 hydroxyl group of isotrichodermol must be acetylated by TRI101 for subsequent pathway genes to function. Despite the importance of this 3-O-acetylation step in biosynthesis, Tri101 is both physically and evolutionarily unrelated to other Tri genes in the trichothecene gene cluster. To gain insight into the evolutionary history of the cluster, we purified recombinant TRI3 (rTRI3), one of the two cluster gene-encoded trichothecene O-acetyltransferases, and examined to determine whether this 15-O-acetyltransferase can add an acetyl to the C-3 hydroxyl group of isotrichodermol. When a high concentration of rTRI3 was used in the assay (final concentration, 50 μM), we observed 3-O-acetylation activity against isotrichodermol that was more than 10⁵ times less efficient than the known 15-O-acetylation activity against 15-deacetylcalonectrin. The rTRI3 protein also exhibited 4-O-acetylation activity when nivalenol was used as a substrate; in addition to 15-acetylnivalenol, di-acetylated derivatives, 4,15-diacetylnivalenol, and, to a lesser extent, 3,15-diacetylnivalenol, were also detected at high enzyme concentrations. The significance of the trace trichothecene 3-O-acetyltransferase activity detected in rTRI3 is discussed in relation to the evolution of the trichothecene gene cluster.

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The photostability of (E)-2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan ((E)-TPMT), the main yellow pigment in salted radish, was studied. First we analyzed the photoproduct generated from (E)-TPMT under longwave UV irradiation. On the basis of NMR spectroscopy, the photoproduct was identified as Z-configurated TPMT, and isomerization from the Z- to the E-form was reversibly induced by Vis-light irradiation. The optimum wavelength for isomerization from the E- to the Z-form was 360–380 nm, and that for isomerization from the Z- to the E-form was 440–460 nm. The E/Z-ratios in the photostationary state under UV- and Vis-light irradiation conditions were approximately 0.95:1 and 26:1 respectively. The (Z)-isomer was more sensitive to light irradiation than the (E)-isomer in the quantum yield measurement. Yellowing was dependent on the ratio of the (Z)-isomer, because the b^* and chroma value rose with increases in the (Z)-isomer by the colorimeters. Hence, it is possible that the formation of the (Z)-isomer contribute to the yellow color of takuan-zuke during long salting and fermentation.

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The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor α (FRα) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FRα, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRα but also RFC and heme carrier protein 1 (HCP1) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFRα, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FRα, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRα, RFC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.

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We investigated the relationship between protein and tryptophan intake and the adverse-effect-level of di(2-ethylhexyl)phthalate (DEHP). Growth retardation of young rats due to DEHP was strengthened by increasing protein level. The addition of tryptophan to the diet caused extreme increases in the nicotinamide formation, but no growth retardation was observed.

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We have reported that propolis suppresses tumor-induced angiogenesis in vivo and in vitro, but antiangiogenic mechanism of propolis at cellular level remains unclear. In this study, we observed that propolis not only inhibited tube formation but also induced apoptosis of endothelial cells. These results suggest that propolis exerts its antiangiogenic effects at least in part through induction of apoptosis.

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Yellowtail meat containing 0 (control), 1% and 2% NaCl weas boiled and stored at 0 °C, and changes in 4-hydroxyhexenal (HHE) and malon aldehyde (MA) contents were analyzed after 0, 1, 2, and 3 d. The HHE contents in all samples increased significantly after 3 d. The MA contents in the NaCl-containing samples were significantly higher than those in the control after storage.

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The extract of the stem bark of Siberian ginseng, Acanthopanax senticosus Harms (ASH), is believed to play a body-coping role in stress through a brain noradrenergic mechanism. The present study was carried out to investigate the effect of ASH on the neuronal activation patterns of c-Fos expression in the rat brain. With ASH administration, c-Fos accumulated in both the supraoptic nuclei (SON) and paraventricular nuclei (PVN), which regulate stress response. Only the caudal regions in the nucleus of the solitary tract (NTS), a locus innervating both the SON and PVN, were activated. Such a neuro-anatomical pattern associated with ASH suggests the possible involvement of these stress-related brain loci.

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A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl β-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4–11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a β-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl β-oligoxylosides.

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During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.

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Expanding the diversity of fungi can lead to the discovery of secondary metabolites that can be applied in the pharmaceutical field. For this purpose, a new simple method using an anthracycline antibiotic, daunomycin (DM), as a selection reagent in isolating slower-growing fungi was established. DM treatment significantly prevented the growth of ordinary faster-growing fungi, thereby fascilitating the selective isolation of slower-growing fungi. In addition, the possibility of isolating rare fungus genera was increased when the DM method was employed. Furthermore, the number of genera obtained by the DM method (56) was larger than that by the Rose Bengal (RB) method (38), and the genera isolated by these methods showed some overlap (20 genera).

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We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.

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A family-4 α-galactosidase Mel4A of Bacillus halodurans was expressed in Escherichia coli and characterized. Recombinant enzyme rMel4A depended on NAD⁺, some divalent cations such as Mn²⁺, and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.

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