Bacteriocins produced by lactic acid bacteria (LAB) are expected to be safe antimicrobial agents. While the best studied LAB bacteriocin, nisin A, is widely utilized as a food preservative, various novel ones are required to control undesirable bacteria more effectively. To discover novel bacteriocins at the early step of the screening process, we developed a rapid screening system that evaluates bacteriocins produced by newly isolated LAB based on their antibacterial spectra and molecular masses. By means of this system, various novel bacteriocins were identified, including a nisin variant, nisin Q, a two-peptide bacteriocin, lactococcin Q, a leaderless bacteriocin, lacticin Q, and a circular bacteriocin, lactocyclicin Q. Moreover, some LAB isolates were found to produce multiple bacteriocins. They were characterized as to their structures, mechanisms of action, and biosynthetic mechanisms. Novel LAB bacteriocins and their biosynthetic mechanisms are expected for applications such as food preservation and peptide engineering.

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Since the discovery of α-glucosidase inhibitors and their inhibitory effects on the digestion of carbohydrates, promising results have been obtained as to the antidiabetic effects of this family of compounds. Antiangiogenic compounds have been identified that suppress tumor growth via a unique mechanism, confirming that such compounds can act as clinically applicable anticancer agents. Lipid peroxidation and lipid glycation have been suggested to play roles in food deterioration and in the pathophysiology of human diseases such as atherogenesis and diabetes, and antioxidative and antiglycative compounds can potentially be used in the prevention of food deterioration as well as to treat disease. On this basis, this review describes studies of α-glucosidase inhibition by mulberry 1-deoxynojirimycin, antiangiogenic effects of rice bran tocotrienol, and membrane lipid peroxidation/glycation and its inhibitors. These studies are ongoing in our work, with an emphasis on analytical techniques.

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Juvenile hormone (JH) I, II and III in the hemolymph of the silkworm, Bombyx mori were quantified by liquid chromatography-mass spectrometry (LC-MS). JHs were treated with methanol and trifluoroacetic acid to convert into JH methoxyhydrines (JH-MHs). The key to the analytical condition for JH-MHs was the addition of 5 µM sodium acetate to the eluting solution. Each JH-MH was observed as the sodium adduct ion with good sensitivity. This improved method enabled the titration of JH I, II and III in hemolymph of the silkworm to be monitored from the 3rd instar through to the early pupal stage. A peak of JH I was observed immediately after ecdysis in the 3rd and 4th instar stages. The JH I titer sharply decreased on day 1 and reached the lowest level before ecdysis, but there was no peak at the beginning of the 5th stadium, and no apparent increase was observed until pupation.

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Silymarin, the seed extract of Silybium marianum, has preventive effects against Alzheimer's disease-like pathogenesis in vivo. We isolated (+)-taxifolin (4) from silymarin as an inhibitor of aggregation of the 42-residue amyloid β-protein. Structure-activity relationship studies revealed the 3',4'-dihydroxyl groups to be critical to the anti-aggregative ability, whereas the 7-hydroxyl group and the stereochemistry at positions 2 and 3 were not important.

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Fluorescent probes are powerful and indispensable tools for imaging RNA in vivo and in vitro. To simultaneously visualize multiple RNA targets in a cell, it is necessary to develop probes which emit fluorescence with different colors by excitation at a single wavelength. We synthesized OMUpy1 and OMUpy2 in this study with a cyanine dye respectively conjugated at their 5' ends. A fluorescent analysis revealed these probes to have yellow or pink fluorescence derived from the cyanine dyes with an extremely large Stokes shift. Three color-coded fluorescent images were also obtained in the presence of target RNAs.

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Bioassay-guided fractionation of a methanol extract of the brown alga, Dictyopteris undulata, led to the isolation of a novel sesquiterpene hydroquinone named zonarenone, together with seven known sesquiterpene hydroquinones, zonarol, isozonarol, yahazunol, zonaroic acid, chromazonarol, isochromazonarol, and 2-(3,7,11-trimethyl-2,6,10-dodecatrienyl)hydroquinone. The structure of zonarenone was elucidated on the basis of spectroscopic information. The isolated compounds, excepting zonaroic acid, showed moderate to high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo and Heterocapsa circularisquama, at a concentration of 1 µg/mL.

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Biochanin A (BCA) is an isoflavone found in red clover possessing multiple pharmacological activities including antimicrobial, antioxidant, and anticancer ones. The present study aimed to assess its hepatoprotective potential at different doses in a carbon tetrachloride (CCl₄)-induced hepatotoxicity model in rats. The effects on hepatic injury were explored by measuring serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. Furthermore, the serum levels of glucose, urea, creatinine, total bilirubin, total proteins, triglycerides, and total cholesterol were determined. The metabolic capacity of the liver was assessed by measuring changes in cytochrome P450 2E1 activity. The underlying mechanisms were substantiated by measuring oxidative stress markers as catalase, superoxide dismutase, glutathione peroxidase, glutathione transferase, glutathione reductase, reduced glutathione, total antioxidant capacity, and lipid peroxidation, as well as inflammation markers such as nitric oxide, inducible nitric oxide synthase, cyclooxygenase2, tumor necrosis factor-α, and leukocyte-common antigen. The results were confirmed by histopathological examination, and the median lethal dose was determined to confirm the safety of the drug. BCA successively protected against CCl₄-induced damage, normalizing many parameters to that of the control group. The study indicates that BCA possesses multimechanistic hepatoprotective activity that can be attributed to its antioxidant, anti-inflammatory, and immunomodulatory actions.

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β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid β-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1 L of culture medium, 2.1 mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 °C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4–9.8 and below 55 °C. Native OsTAGG2 and rOsTAGG2P showed 4.5–4.7-fold higher activities towards salicylic acid β-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid β-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.

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Prokaryotic organelles called magnetosomes allow magnetotactic bacteria to navigate along geomagnetic field lines. In this study, we modified a swimming assay commonly used to assess bacterial motility to develop a new method of assessing magnetotactic motility. By this method, the swimming assay was performed in an artificial magnetic field. Magnetotactic bacteria formed a wedge-shaped swimming halo that elongated parallel to the magnetic field. Magnetotactic motility was qualitatively assessed by comparing halo shapes. We termed this method the magnetic swimming assay. On the magnetic swimming assay, the mamK deletion strain formed a shorter halo than the wild type, indicating that the assay sensitively detects differences in magnetotactic motility. Moreover, we isolated two spontaneous magnetotactic motility mutants using magnetic swimming plates. Our findings indicate that the magnetic swimming assay is a useful method for the sensitive analysis of magnetotaxis phenotypes and mutant screening.

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The present study aimed at a comparative characterization of two distinct extracellular monocrotophos hydrolases, from Penicillium aculeatum ITCC 7980.10 (M3) and Fusarium pallidoroseum ITCC 7785.10 (M4), isolated from agricultural fields. The MCP hydrolases were purified by Sephadex G-100 column and DEAE-Sepharose CL-6B ion-exchange column followed by SDS–PAGE analysis, which showed the presence of two hydrolases, of 33 and 67 kDa respectively. Both enzymes were most active at alkaline pH and were stable over a wide range of temperatures (60–70 °C). Between the strains, the MCP hydrolases from M3 were 2-fold more active than that from M4. Enzyme kinetic studies showed lowest K_m (33.52 mM) and highest V_max (5.18 U/mg protein) for OPH67 of M3 in comparison to the K_m and V_max of the other hydrolases purified from M3 and M4, suggesting that M3 OPH67 was the most efficient MCP hydrolase. To the best of our knowledge, this is the first report of the purification of two distinct extracellular thermostable MCP hydrolases from fungal strains Penicillium aculeatum ITCC 7980.10 and Fusarium pallidoroseum ITCC 7785.10. Owing to its potential MCP hydrolyzing activity, M3 OPH67 can perhaps used directly or in the encapsulated form for remediation of MCP contaminated sites.

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We investigated the role of nitric oxide (NO) in vascular endothelial growth factor (VEGF) expression in the rat placenta. A nitric oxide synthase (NOS) inhibitor, N^G-nitro-L-arginine-methyl ester (L-NAME), was constantly infused into pregnant rats 6–24 h before sacrifice on gestational day (GD) 15.5. NO production declined to about 15% of the control level as monitored by NO trapping and electron paramagnetic resonance spectroscopy. VEGF mRNA expression was temporally decreased by L-NAME, but recovered to normal levels after 24 h of treatment, whereas hypoxia inducible factor (HIF)-1α and induced NOS (iNOS) expression increased. VEGF expression decreased significantly in placental explants after 6 h of co-treatment with L-NAME and lipopolysaccharide, an iNOS inducer. Our data indicate that NO induce VEGF expression in vivo and in vitro in the rat placenta, suggesting that peaked NO production was maintained by a reciprocal relationship between NO and VEGF via HIF-1α.

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Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca²⁺]_cyt oscillation in Arabidopsis.

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In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

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Our purpose was to establish an in vitro model of the human placental barrier based on placenta slice culture and Ussing chamber. The villous morphology, beta-human chorionic gonadotropin (β-hCG), mRNA and efflux function of P-glycoprotein (P-gp), and the permeability of the fluorescent marker were confirmed. The results showed that syncytiotrophoblast cells with abundant endoplasmic reticulum and mitochondria were covered with a dense microvillus in the placenta slice. The β-hCG secretion levels in the Ussing chamber were 274.13 ± 13.52 mIU/mL at 5 h, significantly higher than that in the incubator 95.2 ± 13.14 mIU/mL, and β-hCG continued to secrete for 48 h. P-gp mRNA was expressed in the placenta slice. The Rho123 apparent permeability coefficient (Papp) value from maternal side to the fetal side was 26.34 ± 1.87 nm/s, but it was significantly increased, to 289.55 ± 6.02 nm/s after adding verapamil. The Rho123 efflux value was >2. The fluorescein Papp value was (3.42 ± 0.24) × 10⁻³ nm/s. In contrast, the fluorescein isothiocyanate-dextran (FD70) Papp value was (3.93 ± 0.08) × 10⁻⁵ nm/s. This indicates that the placenta slice in the Ussing chamber had the activity of a placenta, and can act as a valuable in vitro model of placental barrier.

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Chemical compounds are produced every day, many with adverse effects on human health, and hence it is vital to predict the risks to humans simply, rapidly, and accurately. Teratogens have a serious impact on fetal development. This has been studied mainly by phenotypic analysis of experimental animals. However, since phenotypes can vary within different species, we established a new evaluation system based on our recent finding that teratogens influence Hox gene expression in mice. Similarly to the Hox gene expression changes, the expression patterns of several transcription factors involved in development, including the Dlx, Irx, Sall, and T-box families, were altered after 6 h of exposure to retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The expression changes in Dlx4, Dlx6, Irx5, Sall2, Sall3, Sall4, Tbx10, and Tbx22 were linked to teratogen-induced phenotypes, and our results indicate that expression changes in developmental transcription factors can help to predict teratogenic risk.

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Tuliposide A-converting enzyme (TCEA) catalyzes the conversion of 6-tuliposide A to its lactonized aglycon, tulipalin A, in the tulip (Tulipa gesneriana). The TgTCEA gene, isolated previously from petals, was transcribed in all tulip tissues but not in the bulbs despite the presence of TCEA activity, which allowed prediction of the presence of a TgTCEA isozyme gene preferentially expressed in the bulbs. Here, the TgTCEA-b gene, the TgTCEA homolog, was identified in bulbs. TgTCEA-b polypeptides showed approximately 77% identity to the petal TgTCEA. Functional characterization of the recombinant enzyme verified that TgTCEA-b encoded the TCEA. Moreover, the TgTCEA-b was found to be localized to plastids, as found for the petal TgTCEA. Transcript analysis revealed that TgTCEA-b was functionally transcribed in the bulb scales, unlike the TgTCEA gene, whose transcripts were absent there. In contrast, TgTCEA-b transcripts were in the minority in other tissues where TgTCEA transcripts were dominant, indicating a tissue preference for the transcription of those isozyme genes.

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ALG-2 is an EF-hand-type Ca²⁺-binding protein that interacts with a variety of intracellular proteins that possess Pro-rich regions (PRRs) in mammalian cells. IST1 is an endosomal sorting complex required for transport (ESCRT)-III-related charged multivesicular body protein (CHMP)-like protein, but unlike other ESCRT-III proteins, mammalian IST1 has a PRR and a distinctive sequence of Met-Pro repeats. We found that ALG-2 binds to IST1 by Far-Western analysis using biotinylated ALG-2 as probe, and that the Met-Pro repeat sequence is essential for interaction. The results of pulldown assays using Strep-tagged ALG-2 and lysates of cells expressing GFP-fused IST1 proteins indicated that the binding of ALG-2 to IST1 is Ca²⁺-dependent, and that it is enhanced by co-expression with CHMP1 proteins. Moreover, pulldown assays using various mutants of GST-ALG-2 revealed that the ability of IST1 to bind to mutants is different from those of known ALG-2-interacting proteins, suggesting that IST1 binds to ALG-2 by a different mode of recognition.

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The antioxidant activities of Salak plum (Salacca edulis) peel extracts were assessed by 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'–azino-bis(3-ethylbenzothaiazoline)-6-sulfonic acid (ABTS), and ferric reducing ability of plasma (FRAP) assays. The ethyl acetate (EtOAc) fraction was the most potent (DPPH_IC50=2.932 ± 0.030 µg/mL, ABTS_IC50=7.933 ± 0.049 µg/mL, FRAP_EC=7,844.44 ± 40.734). Chlorogenic acid was detected as the marker (1.400 ± 0.102 g/kg). The EtOAc fraction was non-cytotoxic in vero and normal human fibroblast (NHF) cells. It exhibited cellular oxidative prevention and damage treatment at 5–40 µg/mL in NHF cells. Salak plum peel loaded liposome consisting of lecithin and hydrophobically modified hydroxyethylcellulose (HMHEC) was developed and found stable with adequate entrapment efficacy. Thus Salak plum peel was highlighted as a potential ecological antioxidant for health promotion aspects, and for cosmetics.

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Amphibian skin is an abundant repository of bioactive peptides, important components of the defensive system. The variability of the bioactive peptide repertoires of individual species remains unclear. In this study, dark-spotted frogs were collected from Kunming in Yunnan Province, China and Guiyang in Guizhou Province, China to determine whether the bioactive peptides in amphibian skin differ between the two regions. Eight antimicrobial peptides and an antioxidant peptide were identified by screening of cDNA library. Among the identified peptides, three antimicrobial peptides (pelophylaxin-2GY, temporin-1GY, and temporin-1KM) and an antioxidant peptide (antioxidin-PN) are reported here for the first time. Nigrocin-1, nigrocin-2, and pelophylaxin-2 were expressed by frogs in both regions. Pelophylaxin-2GY and temporin-1GY were found only in the frogs from Guiyang, whereas antioxidin-PN, esculetin-1, esculetin-2, and temporin-1KM were found only in those from Kunming. This difference was confirmed by allele-specific RT-PCR. The bioactive peptides expressed clearly varied between these populations of the same species.

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In eukaryotes, cell morphogenesis is regulated coordinately with the cell cycle. In fission yeast, the morphogenesis network MOR (morphogenesis Orb6 network) consists of 5 conserved proteins, Pmo25, Nak1, Mor2, Orb6, and Mob2, and is essential for cell polarity control and cell separation following cytokinesis. Here we show that the conserved leucine-rich repeat protein Lrp1 is required for cell morphogenesis as a newly recognized component of MOR. Lrp1 has 4 leucine-rich repeats in its N-terminus and is a homolog of the budding yeast Sog2, which is a component of the RAM network (regulation of Ace2 activity and cellular morphogenesis). Lrp1 was essential for both cell growth and cell morphogenesis as were the other MOR components. Lrp1 was localized to the SPBs (spindle pole bodies, the yeast equivalent of the animal centrosome) throughout the cell cycle and to the medial ring during cytokinesis. Lrp1 interacted with Nak1 and was important for Orb6 kinase activity. Thus Lrp1 proved to function upstream of Orb6 in cell morphogenesis.

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The methionine salvage pathway (MSP) recycles reduced sulfur from 5-methylthioribose. Here we propose a novel ribose metabolic pathway performed by MSP enzymes of Bacilli. MtnK, an initial catalyst of MSP, had significant ribose kinase activity, with V_max and K_m values of 2.9 µmol min⁻¹ mg of protein⁻¹ and 4.8 mM. Downstream enzymes catalyzed the isomerization of ribose-1-phosphate and subsequent dehydration, enolization, dephosphorylation, and dioxygenation.

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Salicylic acid (SA), yeast elicitor (YEL), and chitosan (CHT) induced stomatal closure in Arabidopsis wild-type and aba2-2 plants, induced stomatal closure in fluridon-treated wild-type plants, and induced stomatal closure in aos mutants. These results suggest that neither endogenous abscisic acid nor endogenous jasmonic acid is involved in SA-, YEL-, or CHT-induced stomatal closure.

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CD36 binds oxidized low-density lipoprotein (oxLDL). A synthetic peptide comprising amino-acid residues 149–168 of mouse CD36 was recently found to bind fluorescence-labeled oxLDL particles. Based on our oxLDL-binding analysis of various synthetic CD36 peptides, we suggest that not only hydrophilic residues (e.g., Lys164 and Lys166) but also hydrophobic ones (e.g., Phe153, Leu158, and Leu161) are critical to binding.

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A wet precipitate is generated in the process of making tofu by adding a coagulant to the basic soymilk ingredient. We investigated the magnesium chloride concentration-dependent change in the precipitate weight. The wet precipitate weight dramatically increased following a short plateau phase at a low concentration of magnesium chloride. It is interesting that this weight slightly decreased following a long plateau phase at a high concentration. These low and high concentrations respectively induced precipitates with a smooth surface and rough surface. The precipitate with a smooth surface had a higher water content than that with a rough surface. These precipitates also had obviously different solubility in various chemical reagents. The different properties indicate that these precipitates were formed by different intermolecular interactions. These results can be utilized to more clearly understand the mechanisms involved in tofu formation.

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Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS–PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment.

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Xanthone compounds in mangosteen (Garcinia mangostana Linn.) fruit have been reported to have biological activities including antioxidative and anti-inflammatory effects, and the major xanthone compounds in mangosteen are α-mangostin and γ-mangostin. The objectives of this research were to quantify and qualify the major xanthones in each part of the mangosteen fruit with and without yellow gum from the point of view of effective utilization of agricultural product. Quantitative evaluation revealed that yellow gum had extremely high amounts of α-mangostin and γ-mangostin (382.2 and 144.9 mg/g on a wet basis, respectively) followed by pericarp and aril. In mangosteen fruit with yellow gum inside, xanthones seemed to have shifted from the pericarp and to have concentrated in a gum on the surface of aril, and there was almost no difference between the amounts of α-mangostin and γ-mangostin in whole fruits with and without yellow gum. Pericarp and yellow gum showed much higher radical-scavenging activity and ferric reducing antioxidant potential than the aril.

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Colostrum is a complex mixture of bioactives that promotes neonate growth. Recently, we have found by in vivo study that skimmed, sterilized, and concentrated bovine late colostrum (SCBLC), obtained from a Holstein herd on days 6–7 after parturition, had an ability to maintain intestinal integrity. In the present study we investigated effects of SCBLC on rat intestinal IEC–6 cell proliferation in vitro. A fraction containing αs1–casein was found to have a robust stimulation effect as compared to other protein fractions from SCBLC and even the αs1–casein fraction from milk from other Holstein herds. Furthermore, the SCBLC αs1–casein molecule demonstrated not only slightly slower mobility on both SDS– and native–PAGE than other bovine milk αs1–caseins, but also a peculiar conformation reminiscent of moltenglobule in the circular dichroism spectrum. These findings may be of relevant to the competence of SCBLC to preserve intestinal integrity.

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Lactoferrin (LF) is an iron-binding glycoprotein contained in milk and other exocrine fluids, and is believed to have multiple biological functions. We investigated the intracellular dynamics of LF taken up by three lines of human enterocytes and the subsequent release of internalized LF by using two-site ELISA and confocal microscopy. LF taken up by Caco-2 cells was kept partially intact within the cells and subsequently released to the medium as degraded fragments of 30–50 kDa. The retention and subsequent release of LF by Caco-2 cells were much more abundant than those of ovalbumin, ovomucoid and lysozyme. Such results characteristic of LF were also similarly observed in C2BBe1 and HT29 cells more markedly. LF was detected as punctate signals and partially colocalized with the lactoferrin receptor, intelectin-1, in the respective cytoplasm and nuclei of Caco-2 and C2BBe1 cells. In contrast, LF within the HT-29 cells was detected as much smaller punctate signals scattered in the cytoplasm.

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The low bioavailability of lipophilic micronutrients is mainly caused by their limited solubilization to an aqueous micelle, which hinders their ability to be taken up by the intestines. Bioaccessibility is the ratio of the solubilized portion to the whole amount ingested. We evaluated in this study the effects of individual fats and oils and their constituents on the bioaccessibility of carotenoids and vitamin E in vegetables by simulated digestion. Various fats and oils and long-chain triacylglycerols enhanced the bioaccessibility of β-carotene present in spinach, but not of lutein and α-tocopherol, which are less hydrophobic than β-carotene. Free fatty acid, monoacylglycerol, and diacylglycerol also enhanced the bioaccessibility of β-carotene present in spinach. In addition to the long-chain triacylglycerols, their hydrolyzates formed during digestion would facilitate the dispersion and solubilization of β-carotene into mixed micelles. Dietary fats and oils would therefore enhance the bioaccessibility of hydrophobic carotenes present in vegetables.

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This study was conducted to investigate the expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, in the smooth muscle of the chicken gizzard. Atrogin-1/MAFbx mRNA expression was detected in the skeletal muscle, heart (cardiac muscle), gizzard (smooth muscle), brain, and liver of chicks, with highest expression in the smooth muscle of the gizzard. The expression of atrogin-1/MAFbx mRNA in the smooth muscle of the gizzard was increased by fasting (24 h), and this increase was reduced by refeeding (2 h). These results indicate that atrogin-1/MAFbx mRNA is highly expressed in the smooth muscle of the chicken gizzard, and that the expression of it is regulated by nutritional conditions.

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The purpose of this study was to determine the effects of chronic renal failure (CRF) on B-group vitamin status using model rats in which adenine-induced CRF. We measured B-groups vitamins in the urine, blood, liver, and kidney. These results showed that renal failure affected the distribution, metabolism, and renal clearance of water-soluble vitamins, and that the effects were different with each vitamin.

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We investigated the anti-obesity effects of dietary D-psicose on adult rats fed a high-sucrose diet. Wistar rats (16 weeks old) that had previously been fed a high-sucrose diet (HSD) were fed HSD or a high-starch diet (HTD) with or without 5% D-psicose for 8 weeks. The food efficiency, carcass fat percentage, abdominal fat accumulation, and body weight gain were all significantly suppressed by dietary D-psicose.

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Arbutin and deoxy arbutin may release hydroquinone under some conditions. We therefore investigated the photostability of arbutin and deoxy arbutin in an aqueous solution. The results revealed arbutin and deoxy arbutin to be photolabile in an aqueous solution. Deoxy arbutin was less stable than arbutin when exposed to UV radiation. The hydroquinone concentration was also increased during the radiation period in both solutions. Benzophenone-4 could clearly improve the photostability of arbutin during the period of UV radiation, but only slightly enhance the photostability of deoxy arbutin.

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Ginsenoside 20-O-β-D glucopyranosyl-20(S)-protopanaxadiol (compound K), a minor ginsenoside, is not found in white raw ginseng, but has better bioavailability than the major ginsenosides in ginseng. Employing commercial enzyme packages for industrial applications, the optimum conditions for enzymatic transformation for the highest content of compound K was explored to enhance the health benefits of ginseng extract. Cytolase PCL 5 was selected from commercial enzyme packages nominated for high β-glucosidase activity. By response surface methodology, the optimal conditions were identified as 78 h of treatment at pH 4.3 at 55.4 °C for 2.068 mg/mL of compound K, showing good agreement with the experimental value.

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To remove nitrogen efficiently from high-concentration organic wastewater, we studied breeding methods using Saccharomyces cerevisiae as a model yeast with improved nitrogen accumulation ability. By DNA microarray analysis under various nitrogen concentrations with two nitrogen sources (peptone and L-asparagine), we obtained 295 commonly overexpressed (over 2-fold) genes and 283 commonly underexpressed (under one-half) genes under nitrogen-starvation conditions. We speculated that overexpression or underexpression recombination of some of these genes might enhance nitrogen uptake. Because a complete collection of nonessential gene deletion strains had been created, we investigated the nitrogen accumulation profiles of underexpressed gene deletion strains. From 256 nonessential gene deletion strains, three (URE2, SNO1, and AVT3) were selected. Strain SUD2 (ure2Δ::kanMX4) improved by 1.2-fold total nitrogen per cell (TN/OD₆₆₀) as compared to the parent strain, S288c. Positive selection of methylamine-resistant mutants to obtain URE2 mutants was useful for improving nitrogen accumulation ability without recombinant techniques.

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Staphylococcus aureus and Enterococcus faecalis employ cyclic peptide-mediated quorum sensing (QS) systems, termed agr and fsr respectively, to regulate the expression of a series of virulence genes. To identify quorum sensing inhibitors (QSIs) that target agr/fsr systems, an efficient screening system was established. In addition to the gelatinase-induction assay to examine E. faecalis fsr QS, the use of an S. aureus agr reporter strain that carries luciferase and green fluorescence protein genes under the agr P3 promoter facilitated the development of a high-throughput screen (HTS) for QSIs. As a result of screening of 906 actinomycetes culture extracts, four showed QSI activity against the agr and fsr systems without growth inhibitory activity. The extracts were purified on a small scale, and three HPLC peaks were obtained with obvious QSI activity. In sum, the established HTS system is a promising strategy for the discovery of anti-pathogenic agents targeting cyclic peptide-mediated QS in Gram-positive pathogens.

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Metabolic pathway engineering of cyanobacteria for the production of industrially important chemicals from atmospheric CO₂ has generated interest recently. Here, we engineered Synechocystis sp. PCC 6803 to produce lactic acid using a lactate dehydrogenase (ldh) gene from various lactic acid-producing bacteria, Lactococcus lactis (ldhB and ldhX), Lactobacillus plantarum (ldhL and ldh), and Lactobacillus rhamnosus (ldhL). The lactic acid was secreted outside the cell using a transporter (lldp) gene from L. plantarum. Expression of each ldh in Synechocystis sp. PCC6803 was ascertained by reverse-transcriptase polymerase chain reaction. Five transformants led to the production of L-lactic acid. Co-expression of lldp with ldhB from L. plantarum or ldhL from L. rhamnosus led to the secretion of lactic acid into the medium at concentration of 0.17 ± 0.02 or 0.14 ± 0.02 mM after 18 d of cultivation.

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The Corynebacterium glutamicum NCgl1221 mechanosensitive channel mediates L-glutamate secretion by sensing changes in membrane tension caused by treatments such as biotin limitation and penicillin. The NCgl1221 protein has an N-terminal domain (1–286 a.a.) homologous to the Escherichia coli MscS and a long C-terminal domain (287–533 a.a.) of unknown function. In order to investigate the role of the C-terminal domain in L-glutamate secretion, we constructed a series of C-terminally truncated mutants of NCgl1221. We found that the N-terminal domain, homologous to E. coli MscS, retained the ability to cause L-glutamate secretion in response to the treatment. Electrophysiological analysis confirmed that the N-terminal domain mediated L-glutamate secretion. 3D homology modeling has suggested that the N-terminal domain of NCgl1221 has an extra loop structure (221–232 a.a.) that is not found in most other MscS proteins. The mutant NCgl1221, deleted for this loop structure, lost the ability to secrete L-glutamate. In addition, we found that mutant NCgl1221 lacking the C-terminal extracytoplasmic domain (420–533 a.a.) produced L-glutamate without any inducing treatment. These results suggest that the N-terminal domain is necessary and sufficient for the excretion of L-glutamate in response to inducing treatment, and that the C-terminal extracytoplasmic domain has a negative regulatory role in L-glutamate production.

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Although lactose is a preferred cellulase inducer in the industrial production of cellulase by Trichoderma reesei, the mechanism of induction is not fully understood. Because sugar transporters might be involved at an early step of induction by oligosaccharides, we sought permeases associated with cellulase induction by lactose. Two such MFS sugar transporters in the T. reesei hyper-cellulolytic PC-3-7 strain, an industrial cellulase producer developed in Japan, were identified in a screening for lactose permeases. Disruption of the genes encoding these two transporters resulted in decreased lactose uptake and delayed growth in lactose culture. Further, the deletion strains produced less cellulase when cultivated on lactose. No substantial differences were observed in cellulase production when PC-3-7 was cultivated in cellulose-based medium. The present work provides evidence that these transporters are critical for cellulase production in lactose culture.

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Fumitremorgins (FTMs), tremorgenic mycotoxins produced by the human pathogen Aspergillus fumigatus, are prenylated indole alkaloids that have been extensively studied in view of their diverse chemical structures and biological activities. Their biosynthetic gene (ftm) cluster was identified on the basis of the genome sequence of A. fumigatus. However, it has been reported that the ftm cluster in genome reference strain Af293 is inactive, which makes complete understanding of the FTM pathway difficult. Hence, we used an FTM-producing strain of A. fumigatus, BM939, to dissect the FTM pathway. Here, we delineate the genetic determinant for the observed defect in the FTM pathway in A. fumigatus Af293. Metabolite profiling and sequence comparison of the two strains revealed a point mutation in ftmD as a possible cause of altered metabolite production in strain Af293. FTM production in Af293 was restored when a DNA fragment containing ftmD from BM939 was introduced. Biochemical analysis indicated that FtmD is a methyltransferase that catalyzes the conversion of 6-hydroxytryprostatin B into tryprostatin A. The mutated FtmD retained enzymatic activity but did not function under physiological conditions, resulting in blockage of the FTM pathway in A. fumigatus Af293.

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Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

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Bioelectrochemical (−0.8 V, −0.3 V, and +0.6 V vs. Ag/AgCl) and non-bioelectrochemical co-cultures of a hydrogenotrophic methanogen and a cellulolytic bacterium were conducted. Unlike non-bioelectrochemical co-cultures, a cathodic reaction (−0.8 V) increased the growth of the hydrogenotrophic methanogen and the cellulolytic bacterium, by 6.0- and 2.2-fold respectively, and increased cellulose degradation. In contrast, anodic reactions (−0.3 V, +0.6 V) influenced them negatively.

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To improve ethanol production from D-lactate, Jen1p, a monocarboxylate-proton symporter, was constitutively expressed in Saccharomyces cerevisiae NAM34-4C. The mutant produced 2.4 g/L of ethanol, approximately 2.4 times higher than that of the wild-type strain. A monocarboxylate/proton symporter gene (JEN1) null mutant was also constructed. It produced 0.19 g/L of ethanol, 5 times lower than that of the wild-type strain.

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D-Ribose and 2-deoxy-D-ribose were oxidized to 4-keto-D-ribonate and 2-deoxy-4-keto-D-ribonate respectively by oxidative fermentation, and the chemical structures of the oxidation products were confirmed to be as expected. Both pentoses are important sugar components of nucleic acids. When examined, purine nucleosidase activity predominated in the membrane fraction of acetic acid bacteria. This is perhaps the first finding of membrane-bound purine nucleosidase.

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Calcineurin (CN) is a Ca²⁺/calmodulin (CaM) dependent serine/threonine protein phosphatase and plays important role in several cellular functions in both higher and lower eukaryotes. Here we report inhibition of CN by linear alkylbenzene sulfonate. The clue to the finding was obtained while identifying the inhibitory material leaching from acrylonitrile butadiene rubber used for packing. Using standard dodecylbenzene sulfonate (C₁₂-LAS), we obtained strong inhibition of CN with a half maximal inhibitory concentration of 9.3 µM, whereas analogs such as p-octylbenzene sulfonate and SDS hardly or only slightly affected CN activity. Three alkaline phosphatases, derived from shrimp, bacteria, and calf-intestine, which exhibit similar enzymatic activities to CN, were not inhibited by C₁₂-LAS at concentrations of up to 100 µM. Furthermore, C₁₂-LAS did not inhibit Ca²⁺/CaM-dependent myosin light chain kinase activity when tested at concentrations of up to 36 µM. The results indicate that C₁₂-LAS is a potent selective inhibitor of CN activity.

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