Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both “inverting” and “retaining” glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.

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Lipooligosaccharide (LOS) is a major immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae. Recent immunochemical studies have found that normal human sera (NHS) contain bactericidal anti-LOS antibodies that bind to the oligosaccharide (OS) moiety of neisserial LOS. Although affinity-purified anti-LOS antibodies can be characterized using 10–100 ng of LOS samples (up to a few tens of pmoles), a more sensitive immunoblotting assay must be established in order to analyze NHS directly and characterize anti-LOS antibodies without affinity purification. We examined analytical PAGE/blot conditions using a 15-well mini gel. For the first time, Western blot detection of LOS at the lower femtomole level was accomplished by both chromogenic and chemiluminescent detection. A model LOS, 15253 LOS, was detected in a low femtomole range (62.5–500 pg, 16–125 femtomole) even with 10 pM of a monoclonal antibody (MAb) 2C7. Furthermore, detection of similar amounts (50–250 femtomole) of neisserial LOSs and Salmonella truncated lipopolysaccharides (LPSs) was also possible with 1:50 and with 1:100 diluted NHS. The results obtained here indicate that the binding of IgG in NHS to the LOS and LPS samples is probably due to their carbohydrate moieties. The detection level accomplished in this study should help not only to further characterize anti-LOS antibodies in blood and body fluids but also to analyze carbohydrate structures that are recognized by them.

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We show that the concentration of total free fatty acids (FFAs) in sake produced by yeast with high productivity of ethyl caproate could be approximated by the concentration of 2 FFAs, caproic and caprylic acids. Measurement of the total FFAs concentration by an enzymatic method proved useful for both estimating the ethyl caproate concentration in sake and also for yeast breeding.

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The content of anthrasesamone C (5), a rare chlorine-containing anthraquinone, in a hairy root culture of Sesamum indicum increased with increasing chloride ion concentration in the culture medium and reached a maximum at 100 mM. However, the amount of anthrasesamone C (5) in the extract obtained from the hairy roots was increased by incubating the extract. This result suggests that anthrasesamone C (5) was produced from an unidentified metabolite by an abiotic process. 2,3-Epoxyanthrasesamone B (1), a precursor for the non-enzymatic formation of anthrasesamone C (5), was isolated from S. indicum hairy roots cultured in a chloride-deficient medium. Its structure was elucidated to be 2,3-epoxy-9,10-dihydroxy-2-(4-methylpent-3-en-1-yl)-2,3-dihydroanthracene-1,4-dione by spectroscopic methods.

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Several flowers of Tulipa gesneriana exhibit a blue color in the bottom segments of the inner perianth. We have previously reported the inner-bottom tissue-specific iron accumulation and expression of the vacuolar iron transporter, TgVit1, in tulip cv. Murasakizuisho. To clarify whether the TgVit1-dependent iron accumulation and blue-color development in tulip petals are universal, we analyzed anthocyanin, its co-pigment components, iron contents and the expression of TgVit1 mRNA in 13 cultivars which show a blue color in the bottom segments of the inner perianth accompanying yellow- and white-colored inner-bottom petals. All of the blue bottom segments contained the same anthocyanin component, delphinidin 3-rutinoside. The flavonol composition varied with cultivar and tissue part. The major flavonol in the bottom segments of the inner perianth was rutin. The iron content in the upper part was less than that in the bottom segments of the inner perianth. The iron content in the yellow and white petals was higher in the bottom segment of the inner perianth than in the upper tissues. TgVit1 mRNA expression was apparent in all of the bottom tissues of the inner perianth. The result of a reproduction experiment by mixing the constituents suggests that the blue coloration in tulip petals is generally caused by iron complexation to delphinidin 3-rutinoside and that the iron complex is solubilized and stabilized by flavonol glycosides. TgVit1-dependent iron accumulation in the bottom segments of the inner perianth might be controlled by an unknown system that differentiated the upper parts and bottom segments of the inner perianth.

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Streptomyces species strain GK18, isolated in Iran, induced deep-pitted lesions on potato tubers, lesions different from the raised lesions induced by the usual scab-causing phytotoxin, thaxtomin. In addition, neither thaxtomin production nor hybridization to its biosynthetic probe was detected for strain GK18, suggesting the production of a different phytotoxin. The active component was extracted with ethyl acetate from culture filtrate of strain GK18, purified by gel filtration and silica gel chromatography, and identified as an 18-membered macrolide, borrelidin, by spectroscopic analysis. The purified borrelidin induced necrosis on potato tuber slices and inhibited the growth of shoots and roots of radish seedlings. This is the first report on the phytotoxicity of borrelidin as a possible causative compound of potato scab disease.

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Bioassay-guided fractionation of a methanol extract of the brown alga, Ishige sinicola, led to the isolation of five algicidal compounds. Their structures were determined to be α-monoglycerides of eicosa-5Z,8Z,11Z,14Z-tetraenoic (arachidonic) acid, octadeca-6Z,9Z,12Z,15Z-tetraenoic acid, linoleic acid and oleic acid, and 1-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol on the basis of spectroscopic data and a comparison with the data in the literature. These glycerolipids showed moderate-to-high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo, Karenia mikimotoi and Alexandrium catenella, at a concentration of 20 μg/mL.

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(R)-10-Methyl-2-tridecanone, the female sex pheromone of the southern corn rootworm (Diabrotica undecimpunctata howardi Barber), was synthesized in 9 steps from methyl (S)-3-hydroxy-2-methylpropanoate in a 15.7% overall yield. Olefin cross metathesis between (R)-6-methyl-1-nonene and 5-hexen-2-one employing Grubbs’ first-generation catalyst was the key step of the synthesis.

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We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9β-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.

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In total, 185 unigenes were identified from 380 clones of postnatal skeletal muscle of Hainan Black goats by suppression subtractive hybridization (SSH) technology. Most of the differentially expressed genes involved energy metabolism and muscle contraction. The expression of 19 genes was analyzed in the longissimus dorsi muscles of 2-, 6-, 12-, 24-month olds, and four gene expression patterns were found by hierarchical cluster analysis. Most genes in first expression pattern belonged to myofibrillar proteins and had higher expression levels at 2 months old; genes of the secondary expression pattern had higher expression levels at 12 months old; tropomyoain 1 (alpha) (TPM1) was classified into the third expression pattern, and its expression level showed decreases tendency as age increased. Tropomyoain 2 (beta) (TPM2) was classified into the third expression pattern, which had the opposite expression pattern against TPM1.

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Archaerhodopsin 4 (AR4), a retinal-containing membrane protein, exhibits a reversed order of proton release and uptake at neutral pH, as compared to the well-known bacteriorhodopsin (BR). In a preceding report, we stated that Triton X-100 solubilized the claret membrane containing AR4 (CM) into monomeric proteins and altered the time order in AR4 at neutral pH. The present study examined the mechanism underlying this phenomenon. We employed a photoelectrochemical cell suitable for observation of the proton pumping behaviors of both the membrane patch and detergent-solubilized proteins over a wide pH range. The pK_a values of the proton release complex (PRC) in the initial state and the M state were determined with this device. The pK_a of PRC of monomeric AR4 decreased to a value lower than 7.0 in the photocycle, allowing early proton release at neutral pH. The pK_a of PRC in the initial state was also strongly affected by solubilization.

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In the fission yeast Schizosaccharomyces pombe, deletion of trt1⁺ causes gradual telomere shortening, while deletion of pot1⁺ causes rapid telomere loss. The double mutant between pot1 and RecQ helicase rqh1 is synthetically lethal. We found that the trt1 rqh1 double mutant was not synthetically lethal. The chromosome end fragments in both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd (helicase dead) double mutants did not enter a pulsed-field electrophoresis gel. Both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd double mutants were sensitive to the anti-microtubule drug thiabendazole. Moreover, the trt1Δ rqh1-hd double mutant displayed RPA foci on the chromosome bridge at high frequency in M phase cells. These phenotypes are very similar to that of the pot1Δ rqh1-hd double mutant, in which recombination intermediates accumulate at the chromosme ends in the M phase. These results suggest that the entangled chromosome ends, most likely recombination intermediates, are present in the M phase in the trt1Δ rqh1-hd double mutant.

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CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0–99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization.

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Soybeans, the world’s leading leguminous crop, establish mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. AM fungi colonize root cortical cells forming arbuscules, highly branched fungal structures. Arbuscules are enveloped by plant-derived periarbuscular membranes through which plants obtain mineral nutrients, particularly phosphate. We searched the soybean genome in silico, and found 14 Pht1 genes encoding phosphate transporters putatively localized on the plasma membranes. Time course analyses involving reverse transcription-PCR indicated that three of these were AM-inducible. GmPT10 and GmPT11 were induced on fungal colonization, while a transcript of GmPT7 appeared in the later stages. The transport activities of GmPT10 and GmPT11 were confirmed by complementation of a yeast mutant. Soybean hairy roots expressing the GmPT10-green fluorescent protein (GFP) or GmPT11-GFP fusion protein under the control of corresponding promoter showed GFP fluorescence on the branch domains of periarbuscular membranes, indicating that active phosphate transport occurred there.

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A galactose specific lectin (CpL) was purified from the Clavaria purpurea mushroom by affinity chromatography. CpL agglutinated only trypsin-treated rabbit erythrocytes. On enzyme linked lectin sorbent assay (ELLSA), the lectin bound with thyroglobulin and asialo bovine submaxillary mucin (BSM). The fine sugar binding specificity of CpL was elucidated using inhibition of hemagglutination and sugar immobilized gold nano-particles (SGNP). The results indicated a preference of CpL towards α-galactosyl sugar chains. Among several monosaccharides and disaccharides assayed for dissociation effect on the SGNP-CpL complex, Galα1-3Gal and raffinose were the best inhibitors. The partial amino acid sequence showed two QXW motifs in CpL and similarity towards members of the ricin B superfamily.

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Bacterial laminaribiose phosphorylase (LBP_bac) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into α-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBP_Eug): LBP_bac was more specific to laminaribiose than LBP_Eug. It showed acceptor specificity in reverse phosphorolysis similar to LBP_Eug. Cloning of the gene encoding LBP_bac (lbpA) has revealed that LBP_bac is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N′-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of lbpA, suggesting that the role of LBP_bac is to utilize laminaribiose generated outside the cell. This role is different from that of LBP_Eug, which participates in the utilization of paramylon, the intracellular storage 1,3-β-glucan.

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Paclitaxel (Taxol), one of the most potent anticancer drugs, is a microtubule-stabilizing compound that inhibits microtubule depolymerization within the cell. The structure of paclitaxel is composed of two key elements, a taxane ring and an N-benzoylphenylisoserine side chain at C-13. A number of natural and artificial compounds with taxane skeletons have been isolated, but almost none of their bioactivities have been evaluated. In this study, we focused on compounds having a taxane skeleton structure and examined their effects on tubulin dynamics. Although none of these compounds had an N-benzoylphenylisoserine side chain, three were found to promote tubulin assembly. On the other hand, one compound inhibited tubluin assembly in a way similar to nocodazole. These compounds exhibited novel structure-activity relationships of taxane compounds.

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Here we report the construction of an alternative episomal vector, pBGP3, which allows the expression of heterologous proteins with N-terminal hexahistidine and myc-epitope tags in Pichia pastoris. To test the usefulness of pBGP3, four cellulases from termites were expressed. Production was confirmed by activity assays and Western blot using anti-c-Myc antibody. Purification was performed by single-step Ni²⁺-affinity chromatography, which confirmed the efficiency of pBGP3.

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The Southern root-knot nematode Meloidogyne incognita causes huge crop losses. We investigated the effects of RNA interference directed against the M. incognita posterior segregation-1 gene. The RNAi resulted in a drastic reduction in the relative amount of pos-1 transcript. Also, it reduced reproductive potential in terms of hatching rate from eggs, probably due to embryonic lethality.

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The AmphDI glycosyltransferase transfers a mycosaminyl sugar residue from GDP onto 8-deoxyamphoteronolide B, the aglycone of the antifungal amphotericin B. In this study the amphDI gene was inactivated in Streptomyces nodosus strains lacking the AmphN cytochrome P450. The new mutants produced 8-deoxy-16-methyl-16-descarboxyl amphoteronolides in high yield. These strains and aglycones should prove valuable for in vivo and in vitro glycosylation engineering.

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Enhanced green fluorescent protein (EGFP) and its yellow variant (Venus) are weakly dimeric under physiological conditions. We designed a simple method to evaluate the dimeric tendency of fluorescent proteins in living mammalian cells. A novel single mutation, A206L, interfering with the hydrophobic interactions of the dimer interface in Venus, contributed to its monomerization, and was as effective as the A206K mutation in this assay.

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We produced a monoclonal antibody (mAb) against N^G,N^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.

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A previous report described the presence of a self-splicing group I intron in a flagellin gene from a thermophilic Bacillus species. Here, we present evidence that the splicing reaction of the flagellin introns is dependent on temperature. Furthermore, a complementation analysis using a Bacillus subtilis flagellin-deficient mutant indicated that the intron-containing flagellin gene significantly restored the motility of the mutant at higher temperatures.

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SecA is an ATP-driven motor for Sec translocase that participates in bacterial protein export and thylakoidal import in plants. We have reported that Cyanidioschyzon merolae, a unicellular red alga, possesses a nuclear-encoded secA(nuc) and a plastid-encoded secA(pt) gene. In this study we found that the amount of SecA(nuc) protein almost quadrupled at high temperature, whereas that of the SecA(pt) protein increased far less. We were also able to determine the localization of both SecAs to the chloroplast by immunofluorescence and immunoelectron microscopy. We suggest that SecA(nuc) has an important role in the chloroplast at high temperatures.

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α-L-Arabinofuranosidase from the hyperthermophilic bacterium Thermotoga maritima (Tm-AFase) is an extremely thermophilic enzyme belonging to glycoside hydrolase family 51. It can catalyze the transglycosylation of a novel glycosyl donor, 4,6-dimethoxy-1,3,5-triazin-2-yl (DMT)-β-D-xylopyranoside. In this study we determined the crystal structures of Tm-AFase in substrate-free and complex forms with arabinose and xylose at 1.8–2.3 Å resolution to determine the architecture of the substrate binding pocket. Subsite −1 of Tm-AFase is similar to that of α-L-arabinofuranosidase from Geobacillus stearothermophilus, but the substrate binding pocket of Tm-AFase is narrower and more hydrophobic. Possible substrate binding modes were investigated by automated docking analysis.

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The protective effects of live Lactobacillus paracasei NFRI 7415 on alcoholic liver disease were investigated. Male Fischer 344 rats were fed a control diet (CD), an ethanol diet (ED) (35.8% of total energy from ethanol), or an ethanol diet containing 20% live Lb. paracasei NFRI 7415 (10⁷ cfu/g) (LD) for 10 weeks. The results indicated that live Lb. paracasei NFRI 7415 reduced the total cholesterol concentration of the plasma and liver in the rats fed the LD. The level of docosahexaenoic acid (DHA; 22:6n-3) in the plasma and liver of the LD group was higher than in the ED group. Chronic alcohol consumption decreased the level of n-3 fatty acid in the plasma and liver of the ED group. These results indicated that live Lb. paracasei NFRI 7415 can adjust the fatty acid composition of the plasma and liver, and that it is possible to decrease liver damage due to chronic alcohol intake.

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The nitric-oxide (NO)-cyclic-guanosine-monophosphate (cGMP) pathway plays a key role in penile erection. Erectile dysfunction (ED) is a complication in male diabetic patients that impacts their quality of 1ife. Recently, Yidiyin, a Chinese herbal decoction, is used to treat diabetic ED, but convincing evidence is lacking, and the potential mechanisms remain uncertain. In the study, diabetic ED patients had low scores on international index of erectile function-5 (IIEF-5), and administration of Yidiyin and hypoglycemic drugs for 16 weeks ameliorated patients’ scores on IIEF-5 more than the hypoglycemic drug alone. Moreover, streptozotocin-induced diabetes severely impaired rats’ erectile function and the activity of the NO-cGMP pathway in the corpora cavernosum, and treatment with Yidiyin for 4 weeks obviously increased the rats’ erectile function, remarkably enhanced the activity of nitric oxide synthase (NOS), and elevated the contents of NO and cGMP. Our findings indicate that Yidiyin improves diabetic ED probably by enhancing the NO-cGMP pathway.

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D-Alanylation of teichoic acid (TA) affects various functions of Gram-positive bacteria, including immunomodulatory effects. We investigated in this study the impact of D-alanine (D-Ala) in TA from Streptococcus thermophilus ATCC 19258^T on the barrier-protecting effect in human intestinal Caco-2 cells. ATCC 19258^T suppressed the tumor necrosis factor-α-induced decrease in transepithelial electrical resistance (TER), an indicator of the barrier function. The D-alanylation of TA in ATCC 19258^T was growth phase- and culture temperature-dependent. Treatment of ATCC 19258^T with Mg²⁺ decreased the dlt mRNA expression and D-Ala content in TA and also abolished the suppressive effect on the TER decrease. Supplementation with L-alanine (L-Ala) to the broth led to an increase of D-Ala in ATCC 19258^T and of the intestinal barrier-protecting effect. Taken together, D-Ala in TA played an important role in the barrier-protecting effect of S. thermophilus in the intestinal epithelium, and these beneficial effects could be enhanced by exogenous L-Ala.

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Actinidia polygama Max. was subjected to supercritical fluid extraction (SFE), and the resulting ethanol extract of marc (SFEM) was subjected to sequential fractionation with various solvents. Each extract and fraction was assayed for anti-inflammatory effect. The ethyl acetate fraction (EtOAc) contained the highest level (70.8% inhibition) of anti-inflammatory activity. In order to identify the active constituents, the EtOAc fraction was further fractionated by silica gel and ODS column chromatography. By activity-guided fractionation, an active ceramide was identified as the anti-inflammatory component, and its structure was determined by NMR and MS analysis. The novel ceramide was named actinidiamide, and was found significantly to inhibit nitric oxide (NO) production (30.6% inhibition at 1 μg/mL) in lipopolysaccaride (LPS)-stimulated RAW 264.7 cells and β-hexosaminidase release (91.8% inhibition at 1 μg/mL) in IgE-sensitized RBL-2H3 cells. Thus the presence of actinidiamide conveys allergy and inflammation treatment ability to A. polygama.

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The antioxidative properties of vanillic acid esters were systematically evaluated by multiple assays to compare with the well-known antioxidants, vanillic acid and Trolox. We first performed assays with the model radicals, DPPH, galvinoxyl and ABTS cation (ABTS^•+) types. Methyl vanillate, ethyl vanillate and butyl vanillate showed stronger activity than Trolox in the ABTS^•+-scavenging assay, but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. In contrast, vanillic acid could quench the three radicals. We then evaluated their antioxidative activities by an ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA), using physiologically relevant peroxyl radicals. Vanillic acid esters and vanillic acid exerted much stronger activity than Trolox in the ORAC assay and OxHLIA. The antioxidative activity by OxHLIA was strongly correlated to the lipophilicity of vanillic acid and its esters. These results indicate that the protective effect of vanillic acid esters against free radical-induced biomembrane damage increased with increasing lipophilicity.

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The objective of this study was to investigate the effects of rice porridge on the texture and viscoelastic properties of bread during storage. Three types of bread, wheat flour bread, 15% rice flour bread, and 15% rice porridge bread, were prepared. After baking and storing the bread for 24 h, 48 h, and 72 h at room temperature, we measured the texture and viscoelastic properties of the bread crumbs by texture profile analysis (TPA) and creep test. The 15% rice porridge bread showed a significantly higher specific volume and maintained softer crumbs than the other two types (p<0.05). It also had a slightly stickier texture than the others. It can be concluded that rice porridge improves the specific volume, texture, and viscoelastic properties of bread crumbs during storage.

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We investigated whether Hyuganatsu orange (Citrus tamurana Hort. ex Tanaka) contains water and acetic-acid soluble substances that increase bone mineral density (BMD) in ovariectomized rats. In in vivo study, femoral BMD can significantly increased. In in vitro study, tartrate-resistant acid phosphatase (TRAP) positive cells significantly decreased. We speculate that Hyuganatsu orange contains biologically active substances other than hesperidin that increase BMD.

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The organic germanium compound, Ge-132, has immune-modulating effects. We evaluated the symbiotic effects of Ge-132 with lactobacilli and oligosaccharide (LB/OS) on the immune responses of mice. The highest fecal IgA levels were observed in the mice receiving a low concentration of Ge-132 with LB/OS for 8 weeks. Our data suggest that LB/OS with a low concentration of Ge-132 stimulated the intestinal immunity.

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Pre-treatment with α-tocopherol (α-Toc) potentiated cytotoxicity induction by benzyl isothiocyanate (BITC). Biochemical events related to apoptosis, such as DNA ladder formation and caspase-3 activation, were also enhanced by α-Toc. These results suggest a significant role of the caspase-3 pathway in apoptosis induction regulated by α-Toc in combination.

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We compared the antioxidative activities of polyphenol metabolites with those of intact functional polyphenols by an assay of the oxygen radical absorbance capacity (ORAC). The metabolites of ellagitannin geraniin, chlorogenic acid, and (−)-epigallocatechin gallate displayed more potent antioxidative activity than their respective original compounds. Our findings suggest that these metabolites may play important roles as biological antioxidants after their consumption.

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Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI⁻ concentration in the assay mixture after ultrafiltration.

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Acremonium cellulolyticus CF-2612 is a cellulase hyper-producing mutant that originated from A. cellulolyticus Y-94. In this study, we isolated a uracil auxotroph (strain CFP3) derived from CF-2612, and cloned a wild-type pyrF gene encoding orotate phosphoribosyl transferase (OPRTase) from Y-94. OPRTase activity was not detected in strain CFP3, which had one nucleotide substitution in its pyrF gene. The wild-type pyrF gene restored the defective growth of CFP3 on uracil-free medium, and PCR and Southern analyses revealed that wild-type pyrF was integrated into the genome. These results indicate that our transformation system for A. cellulolyticus with the pyrFgene as a selection marker was successful.

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In single-chamber microbial fuel cells (SC-MFCs), oxygen molecules diffuse through air cathodes into electrolytes and compete against anodes in accepting electrons. In this study, we constructed multiple gene-knockout mutants for terminal oxidases (SO4607, SO2364, and SO3286) in Shewanella oneidensis MR-1 and examined their abilities to generate electric currents in SC-MFCs. Although single-knockout mutants generated levels of current similar to that of the wild type (WT), an SO4607/SO2364 double-knockout mutant (DO) generated 50% higher current than WT. A triple-knockout mutant did not grow in SC-MFC. The Coulombic efficiencies in SC-MFC were, however, not substantially different between WT and DO. In aerobically grown DO cells, the transcription levels of the genes involved in extracellular electron transfer (mtrC and crp) were increased compared to those in WT cells. These results suggest that suppression of aerobic respiration activates the expression of genes related to the extracellular electron transfer and increases the electric output from SC-MFCs.

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Methylotrophic yeasts, which can utilize methanol as sole carbon and energy source, are exposed to two toxic metabolic intermediates, formaldehyde and hydrogen peroxide, during growth on methanol. Here we report that Msn5p, an importin-β family nuclear exporter, participated in the formaldehyde resistance mechanism but not in the hydrogen peroxide resistance mechanism in Candida boidinii. Disruption of the MSN5 gene in this yeast caused retardation of growth on formaldehyde-generating growth substrates such as methanol and methylamine, but the expression levels of the methanol-metabolizing enzymes did not fall. The Msn5p-depleted strain was sensitive to formaldehyde but not to hydrogen peroxide. Furthermore, a yellow fluorescent protein-tagged Msn5p was diffuse in the cytoplasm of C. boidinii when the cells were treated with high concentrations of formaldehyde or ethanol, but was predominantly associated with the nuclei following treatment with hydrogen peroxide.

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Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS–PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.

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The marine sediment-derived fungus Penicillium commune QSD-17 was re-investigated and cultured on rice solid medium. Two new compounds, isophomenone (1) and 3-deacetylcitreohybridonol (2), together with seven known derivatives (3–9), were identified. Their structures were determined by spectroscopic analysis.

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We have reported that lactobionic acid is produced from lactose by Acetobacter orientalis in traditional Caucasian fermented milk. To maximize the application of lactobionic acid, we investigated favorable conditions for the preparation of resting A. orientalis cells and lactose oxidation. The resting cells, prepared under the most favorable conditions, effectively oxidized 2–10% lactose at 97.2 to 99.7 mol % yield.

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