Cytosolic and membrane-associated proteins prepared from rice cells were separated and compared by two different 2D-PAGE methods, isoelectric focusing (IEF)/SDS-PAGE and nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. Although IEF/SDS-PAGE of the cytosolic proteins showed sufficient resolution, some mitochondrial and basic microsomal membrane-associated proteins were weakly or hardly detectable on the 2D gel. High-quality and -quantity separation of the organelle membrane-associated proteins was accomplished by NEPHGE/SDS-PAGE, the advantage of this method being more critical in tightly membrane-bound proteins that were unwashable with NaCl. These results indicate that NEPHGE/SDS-PAGE is a useful tool for the proteomic analysis of rice membrane-associated proteins.

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  All of the four stereoisomers of 3,12-dimethylheptacosane were synthesized from the enantiomers of citronellal. (Z)-9-Pentacosene and (Z)-9-heptacosene were also synthesized. These hydrocarbons are the characteristic components of the cuticular hydrocarbons of the queen of the ant, Diacamma sp.

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  All the eight stereoisomers of (+)-preussin (1b), an antifungal agent inhibiting the growth of fission yeast and human cancer cells, were synthesized in two steps by non-stereoselective reactions and chromatographic separation, starting from L- and D-N-protected-phenylalaninal (2). Their bioassay revealed all of the stereoisomers to be almost equally bioactive.

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  The asymmetric biohydrolysis is described of o-nitrostyrene oxide with high selectivity by whole cells of Aspergillus niger CGMCC 0496. Both the epoxide and diol could be obtained in a high state of optical purity (over 98%). Product inhibition was found when using a high ratio of substrate to cells.

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  A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC₅₀ value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).

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  6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.

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  The antifungal activities of rye seed chitinase-a (RSC-a, class I) and -c (RSC-c, class II) were studied in detail using two different bioassays with Trichoderma sp. as well as binding and degradation experiments with the cell walls prepared from its mycelia. RSC-a inhibited more strongly the re-extension of the hyphae, containing mainly mature cells, than RSC-c did. Upon incubation of the fungus with fluorescent chitinases, FITC- labeled RSC-a was found to be located in the hyphal tips, lateral walls, and septa, while FITC-labeled RSC-c was only in the hyphal tip. RSC-a had a greater affinity for the cell walls than RSC-c. RSC-a liberated a larger amount of reducing sugar from the cell walls than RSC-c did. These results inferred that RSC-a first binds to the lateral walls and septa, consisting of the mature cell walls, and degrades mature chitin fiber, while RSC-c binds only to the hyphal tip followed by degradation of only nascent chitin. As a result, RSC-a inhibited fungal growth more effectively than RSC-c. Furthermore, it was suggested that the chitin-binding domain in RSC-a assists the antifungal action of RSC-a by binding to the fungal hypha.

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An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp. I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100. Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa. The optimum temperature for the activity was 75°C, and the activity was very stable at temperatures below 70°C. The active pH range of this lipase was 1.9-7.2, and the activity was stable at pH below 7.1. The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates. The lipase was very stable in the presence of various organic solvents at a concentration of 40%. Although the N- terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.

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  The DNA sequence of the thermostable chitosanase TCH-2 gene from Bacillus coagulans CK108 showed a 843-bp open reading frame that encodes a protein of 280 amino acids with a signal peptide corresponding to 32 kDa in size. The deduced amino acid sequence of the chitosanase from Bacillus coagulans CK108 has 61.6%, 48.0%, and 12.6% identities to those from Bacillus ehemensis, Bacillus circulans, and Bacillus subtilis, respectively. C-Terminal homology analysis shows that the enzyme belongs to the Cluster I group. The size of the gene was similar to those from mesophiles of the Cluster I group with regard to higher preference for codons ending in G or C. The recombinant chitosanase was electrophoretically purified to homogeneity by only two steps with column chromatography. The half-life of the enzyme was 40 min at 90°C. The purified protein was also highly stable, retaining above 50% residual activities during treatment with denaturants such as urea (8 M) and guanidine•HCl (4 M) at 37°C for 30 min. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, producing the tetramer as a major product.

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  A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.

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  Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1).¹ The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000±1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K₃ were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.

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  Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57^Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57^Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.

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  α-Glucan L-FV-II and β-glucan L-FV-I were shown to co-exist in the extract of fruiting bodies of Lentinus edodes with aq. 5% NaOH/0.05% NaBH₄ in previous work. Water-insoluble α-(1→3)-D-glucan (L-FV-II) was treated with sulfur trioxide-pyridine complex at 25°C to synthesize the water-soluble sulfated derivative SL-FV-II with a degree of substitution (DS) 1.1 in non-selective sulfation. The weight-average molecular weight (M_w) of sulfated glucan SL-FV-II is 57% of that of the original α-glucan L-FV-II. The α-glucan administered by gavaging at a dose of 50 mg/kg of body weight to BALB/C mice having implanted solid Sarcoma 180 was effective at an inhibition rate of 42%. In vitro experiments using human and murine tumor cell lines showed that SL-FV-II had antiproliferation activity at the concentration of 20 μg/mL towards four tumor cell lines. The sulfated α-(1→3)-D-glucan had potent antiproliferation action (52%) on human MCF-7 breast carcinoma cells.

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  The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from α-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.

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  To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)₂ almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)₆ to (GlcNAc)₃, while ChiA exclusively generated (GlcNAc)₂ and (GlcNAc)₄. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.

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  Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD_ChiC) differs from those of the plant enzymes. ChBD_ChiC and the catalytic domain (CatD_ChiC), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD_ChiC binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD_ChiC resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD_ChiC itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD_ChiC in trans.

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  The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-Δ²-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-Δ²-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5α harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5α carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-Δ²-thiazoline-4- carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-Δ²-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.

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  The transcriptional regulatory region of Elf-1 was analyzed by the combination of a yeast one-hybrid system and site-directed mutagenesis. This analysis enabled us to map an activation region between 85-175 of Elf-1.

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  A diphenyl α-aminoalkylphosphonate derivative, which is an irreversible inhibitor of chymotrypsin-like serine proteases, was immobilized on cyanogen bromide-activated Sepharose, and the selective binding of chymotrypsin to the obtained inhibitor-gel was evaluated using batch and column methods. Complete removal of chymotrypsin in an aqueous solution was done using the column method, while partial removal was done using the batch method.

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  The mouse Na⁺/taurocholate cotransporting polypeptide transiently expressed in COS-7 cells caused sodium-dependent uptake of [³H]taurocholic acid with K_m and V_max values of 18 μM and 102 pmol/mg protein/min, respectively. This K_m value is comparable to that for rat NTCP and higher than that for human NTCP. Substrate specificity was evaluated by measuring inhibitory effects of unlabeled bile acids on [³H]taurocholic acid transport.

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  The hydrolytic reactions of Bombyx mori 65-kDa chitinase with the short substrates, N-acetylchitooligosaccharides, were analyzed by HPLC. Analysis of the hydrolyzed products showed that the newly produced oligosaccharides are all β anomers, suggesting that, similar to other family 18 glycosyl hydrolases, the 65-kDa chitinase acts in the retaining mechanism. Furthermore, the enzyme cleaves the N-acetylchitooligosaccharides mainly at the linkage between the second and the third GlcNAc moieties from the non-reducing end, while the other sites were cleaved in smaller proportions. Moreover, the initial reaction rates of the enzyme with the longer N-acetylchitooligosaccharides were higher than those with shorter ones. These results suggest that the enzyme is an endo-cleaving type and more efficient on the longer substrates.

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  The structural gene for NADP+-dependent serine dehydrogenase [EC 1.1.1.-] from Agrobacterium tumefaciens ICR 1600 was cloned into Escherichia coli cells and its complete DNA sequence was analyzed. The gene encodes a polypeptide containing 249 amino acid residues. The enzyme had high sequence similarity to short-chain alcohol dehydrogenases from bacteria and unknown proteins of Haemophilus influenzae, Escherichia coli, and Saccharomyces cerevisiae.

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  Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine γ-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.

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  The chitinase A (chiA) gene from Enterobacter sp. G-1 and the chitosanase A (choA) gene from Matsuebacter chitosanotabidus 3001 were expressed separately and simultaneously in the yeast Schizosaccharomyces pombe. The chiA gene was placed under the transcriptional control of the nmt1 promoter with the glutathione S transferase (GST), and the choA gene was expressed by the human cytomegalovirus (hCMV) promoter with the P factor secretion signal (P3). The expressed proteins of ChoA and GST-ChiA were enzymatical active and were detected as 34-kDa and 80-kDa, respectively, by Western blot analysis. The transformant that expressed the choA gene was able to secrete ChoA efficiently into the culture medium with the specific activity of 102.36 U/mg protein. When the chiA gene was expressed in S. pombe, yeast cells grew slowly and cells became elongated, but when the choA gene was expressed, cells became swollen. Expression of both the chiA and the choA genes resulted in elongated and fat cells.

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  An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5′-phosphate by column chromatography.

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  Soluble soybean polysaccharides (SSPS) extracted from soybean cotyledons are acidic polysaccharides, and exhibited a pectin-like structure. After digesting galacturonan with polygalacturonase, two novel galacturonan (GN) fragments, which were directly linked to xylosyl oligosaccharides, were obtained. One consisted of (β-D-Xyl)₇ branched at the C-3 site of 1,4-linked (α-D-GalA)₄, and the other consisted of (β-D-Xyl)₄ branched at the C-3 site of 1,4-linked (α-D-GalA)₃.

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  We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.

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  The hypoglycemic effect of an exo-polymer produced from a submerged mycelial culture of Lentinus edodes was investigated in streptozotocin-induced diabetic rats. The administration of the exo-polymer (200mg/kg BW) reduced the plasma glucose level by as much as 21.5%, and increased plasma insulin by 22.1% as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively. Gel chromatography of the exo-polymer revealed a single peak which is likely to have been a glycoprotein with a molecular weight of 52kDa and was found to contain 83.5% carbohydrate and 16.5% protein. The Sugar and amino acid compositions of the exo-polymer were analyzed in detail.

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  To clarify the biosynthetic pathway to 2-phenylethanol (2), the deuterium-labeled putative precursor, [²H₈]L-phenylalanine ([²H₈-1]), was fed to the flowers of Rosa ‘Hoh-Jun’ and R. damascena Mill. throughout maturation, ceasing feeding at the commencement of petal unfurling and at full bloom. Based on GC-MS analyses, [²H₈]-1 was incorporated into both 2 and 2-phenylethyl β-D-glucopyranoside (3) when the flowers were fed until full bloom, whereas no such incorporation into 2 was apparent when feeding was ceased earlier. In both species of rose, the labeling pattern for 2 was almost identical to that for 3, and indicated the presence of [²H₆]-, [²H₇]- and [²H₈]-2, and [²H₆]-, [²H₇]- and [²H₈]-3. This may be ascribed to the equilibrium between 2 and 3. The labeling pattern for 2 and 3 also indicated that these compounds were produced from 1 via several routes, the route involving phenylpyruvic acid being the major one.)

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  The Th1 and Th2 preference induced by cells from the Peyer's patch (PP) and spleen (SPL) with various doses of an antigen was examined. The same splenic T cell receptor-transgenic CD4⁺ T cells were first incubated with PP or SPL cells in the presence of various doses of an antigen, and the cytokine response was observed after secondary stimulation. A Th2-type pattern was only obtained for primary stimulation at 10 μM of the antigen with PP cells, whereas a Th1 pattern was induced at both higher and lower concentrations. SPL cells in the presence of 0.1 to 1 μM of the antigen induced the secretion of Th2-type cytokines. Ten and 100 μM of the antigen plus SPL cells did not induce the release of a large quantity of cytokines. PP cells induced a different cytokine pattern at the antigen concentration that induced a similar level of T cell proliferation with SPL cells. Our findings suggest that the antigen-dose dependent development of Th1/Th2 cells is differentially modulated by the antigen-presentation function of cells in PP and SPL.

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  The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 μg/ml.

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  To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-β-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H₂O₂)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H₂O₂ in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H₂O₂.

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  The antihypertensive effect of Brand's Essence of Chicken (BEC), a popular chicken extract used as a traditional health food, was examined with stroke-prone spontaneously hypertensive rats (SHRSPs). The animals were maintained from 6 to 25 weeks of age on drinking water with or without BEC. The BEC-fed group showed a significant reduction in the development of hypertension when compared with the control animals. The levels of blood urea nitrogen and plasma creatinine in the BEC-fed group were significantly lower than those in the control group, suggesting that the renal glomerular function had been improved by the daily administration of BEC. It thus seems likely that BEC would be useful as a prophylactic treatment against the development of hypertension and renal injury.

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  Opioid peptide hemorphin-5 (YPWTQ) was released from human hemoglobin by the action of pancreatic elastase. V-hemorphin-5 (VYPWTQ) was also released under the same conditions. The amount of hemorphin-5 was about 1/10 that of V-hemorphin-5. This is the first report showing release of hemorphin-5 only by the action of one enzyme.

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  We re-examined the methods used in an adjustable-current swimming pool for evaluating the endurance capacity of mice to improve the sensitivity and reproducibility. We found that the BALB/c strain was most suitable to minimize the variation in time taken to swim to the point of fatigue. We found that precise adjustment of the apparatus and the use of three primary swimming trials to select mice before a study made the swimming time more uniform. This procedure was used to establish a more precise evaluation protocol.

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  Strain YY529, capable of producing some anti-ice-nucleating materials (ANM), was isolated from the surface of a camphor leaf. Strain YY529 was identified as Bacillus thuringiensis from its characteristics and taxonomy; the optimum temperature and pH for producing these ANMs were 30°C and 7.0, respectively. One of the ANM with the highest activities among them was purified from the culture. The molecular weight of the ANM was approximately 130 kDa based on a gel filtration analysis. We confirmed that this ANM was a polysaccharide based on the results of the treatment with a mannosidase and the molish reaction. In addition, the LCMS analysis showed that this anti-ice-nucleating polysaccharide (ANPS) had the polyacetyl-D-glucosamine moiety in its structure. Furthermore, this ANPS showed its ability as a non-freeze agent in a preservative solution for the cryopreservation of cock liver. This is the first report of ANPS as a novel ANM from Bacillus thuringiensis YY529.

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  An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH₂OH, NaN₃, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl- tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1^T.

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  A bacterial strain F-5-2, isolated from soil and identified as Klebsiella pneumoniae, removed NH₄⁺ completely in 24 h of aerobic cultivation in a medium containing 1 mg/ml of NH₄NO₃. However, 70% of the NO₃⁻ originally provided remained. When 100 μM Fe²⁺ was added to the medium, both NH₄⁺ and NO₃⁻ were removed simultaneously and completely from the culture within 6 h of incubation. In addition, the amount of MoO₄⁻ in the medium markedly affected the bacterial cell growth and utilization of NH₄⁺ and NO₃⁻. The bacterium could remove 4 mg/ml of NH₄NO₃ completely in 48 h of aerobic cultivation in a medium containing 100 μM Fe²⁺ and 0.8 pM MoO₄²⁻. The total nitrogen in the culture containing its cells was decreased to 14% of that in the NH₄NO₃ originally provided. GC-MS analysis demonstrated that N₂ was generated from the nitrogen atoms of both NH₄⁺ and NO₃⁻.

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  Fungal denitrification is a dissimilating metabolic mechanism for nitrate and was first described in Fusarium oxysporum. Here we investigated regulatory systems of expression of CYP55, which encodes cytochrome P450 (P450nor) and is essential for the fungal denitrification. Promoter-reporter analysis of F. oxysporum CYP55 using Escherichia coli β-galactosidase showed that the region between nucleotides −526 and −515 was critical for induction by nitrate. It contained a nucleotide sequence similar to the binding consensus sequence of the pathway-specific transcriptional factor NirA, which induces expression of the nitrate-assimilatory genes of Aspergillus nidulans in the presence of nitrate. This indicates that expression of the nitrate dissimilatory gene (CYP55) is concomitantly regulated with the nitrate-assimilatory genes. The deletion studies also indicated that the nucleotide sequence between −118 and −107, which was similar to the binding consensus of the yeast Rox1p, which represses the anoxic genes under aerobic conditions, was responsible for repression of CYP55 under aerobic conditions. These results indicate that the fungus adapts to the denitrifying conditions by a combination of NirA- and Rox1-like transcription factors.

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  β-Phenylethylamine (PEA) induced an increase in cytosolic free calcium ion concentration ([Ca²⁺]_c) in Saccharomyces cereviseae cells monitored with transgenic aequorin, a Ca²⁺-dependent photoprotein. The PEA-induced [Ca²⁺]_c increase was dependent on the concentrations of PEA applied, and the Ca²⁺ mostly originated from an extracellular source. Preceding the Ca²⁺ influx, H₂O₂ was generated in the cells by the addition of PEA. Externally added H₂O₂ also induced a [Ca²⁺]_c increase. These results suggest that PEA induces the [Ca²⁺]_c increase via H₂O₂ generation. The PEA-induced [Ca²⁺]_c increase occurred in the mid1 mutant with a slightly smaller peak than in the wild-type strain, indicating that Mid1, a stretch-activated nonselective cation channel, may not be mainly involved in the PEA-induced Ca²⁺ influx. When PEA was applied, the MATa mid1 mutant was rescued from α-factor-induced death in a Ca²⁺-limited medium, suggesting that the PEA-induced [Ca²⁺]_c increase can reinforce calcium signaling in the mating pheromone response pathway.

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  The relationship between conidial enzymes of Penicillium expansum and spore germination was examined. The activities of xylanase and pectinase, but not of cellulase and amylase, were detected in the conidia. The levels of xylanase and pectinase were greatly enhanced by xylan and pectin as respective carbon sources in the basal medium. No conidia germinated in the basal medium without a carbon source. The type of carbon source and the enzyme levels of the conidia did not affect the rate of germination. However, a relationship was found between the enzyme levels and the elongation of the germ tubes.

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  Termitomyces-related symbiotic basidiomycetes in the nests of fungus-growing termites (Macrotermitinae) of several genera in Thailand were cultivated and analyzed phylogenetically based on the DNA sequence of nuclear ribosomal RNA genes. The relationships of the symbiotic fungi with host termites and their locality were apparently complex, supporting intricate mechanisms for the termites to acquire the symbionts.

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  The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45°C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis σ^A-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.

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  A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45°C, and its activity was stimulated in the presence of 5 mM CaCl₂, BaCl₂, or MgCl₂. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both haparin and HS, similar to heparinase II from Flavobacterium heparinum.

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