“Regulation and Functional Relevance of Milk Fat Globules and Their Components in the Mammary Gland” by Naohito AOKI, Bioscience, Biotechnology, and Biochemistry , Vol.70, No.9, pp. 2019-2027, September 2006.
This manuscript was canceled on October16, 2013 because it was found containing a number of serious ethical problems.

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The rhizome of Boesenbergia pandurata Schult. was found to possess potent antioxidant activity in a rat brain homogenate model. Bioassay-guided isolation of the active compounds from a CH₂Cl₂–MeOH (1:1) extract led to the isolation of 5-hydroxy-7-methoxyflavanone, panduratin A, 5,7-dihydroxyflavanone, 2′,6′-dihydroxy-4′-methoxychalcone, 2′,4′-dihydroxy-6′-methoxychalcone, and 4-hydroxypanduratin A. Panduratin A, 4-hydroxypanduratin A, and 2′,6′-dihydroxy-4′-methoxychalcone were also found to exert neuroprotective effects.

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Cyanamide (NH₂CN) is found in nature, although it has long been recognized as an industrial product. Distribution of cyanamide in the plant kingdom was investigated using a direct quantitative determination method to detect and measure cyanamide by stable isotope dilution gas chromatography–mass spectrometry (the SID–GC–MS method). The SID–GC–MS method proved to be a robust way to quantify cyanamide contents in the extracts of 101 species of herbaceous plants. The average recovery of cyanamide from all plants tested was 55.6±20.3%. Vicia villosa and V. cracca contained cyanamide at 369–498 μg/gFW and 3,460–3,579 μg/gFW respectively, while the other 99 species contained no detectable cyanamide (<1 μg/gFW). This result suggests that distribution of cyanamide in the plant kingdom is limited and uneven.

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Plants exhibit various defense responses after recognizing elicitor molecules derived from pathogenic microorganisms and host plants. In this study, we developed an improved colorimetric assay for quantifying the generation of H₂O₂ in plant cells, one of the defense responses, to evaluate elicitor activity quantitatively. H₂O₂ is detected using a dye, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), which can be measured by conventional spectrometers in a highly sensitive and quantitative manner. Using this method, we successfully measured the elicitor activity of flagellin-derived peptides in cultured tobacco cells, and identified several structural features of the peptides important for the elicitor activity. The results suggest that the structural factors required for expression of the elicitor activity differ slightly among plant species. The efficient and sensitive assay developed in this study should be useful not only for studying structure-activity relationships, but also for the screening of novel compounds that can induce defense responses.

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Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine are known as insect-produced plant volatile elicitors. The absolute configuration of the hydroxylinolenoyl moiety of volicitin from three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, was determined to be all 17S in high enantiomeric excess. When treated with 30 pmol of (17S)- and (17R)-volicitin, corn seedlings were induced to release volatiles, there being no significant difference in the amount released between the two isomers. On the other hand, N-linolenoyl-L-glutamine was only about 30% as active as volicitin. Among several synthesized N-linolenoylamino acid conjugates, only the L-glutamine conjugate induced the emission of volatile organic compounds. These results show that the L-glutamine moiety of volicitin played a more critical role than the hydroxyl moiety, although both moieties affected the elicitor activity inducing the release of volatiles.

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A new lactarane sesquiterpene, 1,2-dehydrolactarolide A (1), together with lactarorufin A (2), 3-O-ethyllactarolide A (3) and 3-O-ethyllactarolide B (4), was isolated from a mushroom of the Russulaceae family, Lactarius vellereus. Additionally, two lactarane sesquiterpenes, lactarorufin A (2) and lactarolide A (5) were isolated from L. subpiperatus. 1,2-Dehydrolactarolide A (1) showed promotional activity (152% at 3.6×10¹ μM) toward radicle elongation in lettuce seedlings.

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Sphingomonas spp. of α-proteobacteria often play a role in assisting the development of microfloral communities under adverse soil conditions. Using a Frateuria sp. as an indicator for bacterial growth assay, we investigated the bacterial growth-promoting factor in the culture fluids of Sphingomonas sp. EC-K085. This factor was successfully isolated and identified as linear (R,R,R,R)-3-hydroxybutyrate tetramer (HB4), having a hydroxy-end and a carboxy-end group. When 28 μg of HB4 was charged on a paper disc, impregnated Frateuria sp. cells in modified Winogradsky agar medium exhibited a promoted cell growth to form a clear colony emerging zone after a 2-day incubation.

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Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca²⁺ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.

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The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

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We characterized a new cytochrome P450 monooxygenase (P450), CYP724B2, from tomato (Lycopersicon esculentum). CYP724B2 showed 42% and 62% amino acid sequence identity with Arabidopsis DWARF4/CYP90B1 and rice DWARF11/CYP724B1 respectively. Functional assay of CYP724B2 heterologously expressed in insect cells revealed that CYP724B2 catalyzes C-22 hydroxylation of campesterol, indicating that CYP724B2 is a C-22 hydroxylase. We also isolated a tomato CYP90B homolog (CYP90B3) and found that CYP90B3 is a C-22 hydroxylase as well. CYP724B2 and CYP90B3 showed substrate specificities similar to each other toward the biosynthetic intermediate compounds from campesterol to campestanol. Campesterol was the best substrate, and (24R)-ergost-4-en-3-one was also metabolized to the C-22 hydroxylated product to some extent. On the other hand, the P450s catalyzed C-22 hydroxylation of (24R)-5α-ergostan-3-one and campestanol at a trace level, indicating that the compounds after C-5α reduction are poor substrates of CYP724B2 and CYP90B3. In addition, cholesterol (C₂₇ sterol) and sitosterol (C₂₉ sterol) were also converted to C-22 hydroxylated products by the P450s. Furthermore, CYP724B2 and CYP90B3 genes were ubiquitously expressed, and their transcript levels were down-regulated by the exogenous application of brassinolide. These findings strongly suggest that CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.

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Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E₂ and PGF_2α. TNF-α preferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE₂ and PGF_2α. These results indicate that endogenous PGE₂ and PGF_2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.

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The HPLC/MS system, in which a monolithic silica capillary column is directly connected to an electronspray-ionization mass spectrometer, showed superior performance at high mobile phase linear velocity. A two-dimensional (2D) HPLC/MS system was established, using an ion-exchange particle-packed capillary column at the first dimension and a monolithic silica capillary column at the second dimension. In an analysis of tryptic fragments from bovine serum albumin, an 81% sequence coverage, obtained by the 2D-HPLC/MS system, increased by 23% as compared to a 1D-HPLC/MS system. This 2D-HPLC/MS system using a monolithic silica capillary column should be useful for enhancing sequence coverage of tryptic fragments in proteomics.

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Potato (Solanum tuberosum) plants are rich in 9-lipoxygenase, which converts linoleic acid and α-linolenic acid to 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9-HPOD) and 9S-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid (9-HPOT) respectively. The allene oxide synthase (AOS) involved in 9-HPOD/9-HPOT metabolism in potato, however, has not been characterized in detail. We cloned a cDNA encoding a novel AOS from potato sprouts by reverse transcriptase-PCR based on a partial sequence in the EST database. This AOS was successfully expressed in the yeast Pichia pastoris, and purified using Ni-NTA resin. The recombinant enzyme metabolized 9-HPOD, 9-HPOT, 13-HPOD, and 13-HPOT with reaction efficiencies of 2.5×10⁷, 1.0×10⁷, 2.5×10⁶, and 7.6×10⁶ M⁻¹ s⁻¹ respectively. The α-ketol formed from 9-HPOD was composed mainly of the 9R-enatimomer (90%). Besides sprouts, the mRNA of this AOS was detected in buds, flowers, and stems, but not in leaves, tubers, or roots of mature plants, suggesting that this enzyme has a tissue-specific function.

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No satisfactory treatment is currently available for metastatic malignant melanoma. Recently, the flavonoid quercetin was suggested as a potential treatment due to its anti-tumorogenic properties. Some of these properties appeared to correspond to those published for UVB irradiation. To determine quercetin’s long-term effects, type of apoptosis, and shared properties with UVB, we exposed Mel-Juso, M14, and G361 human melanoma cell-lines to a large range of quercetin or UVB doses, 20–400 μm and 25–1000 mJ/cm² respectively. Apoptosis was measured for 4 consecutive d by flow cytometry and cell viability was studied by colony-forming assay. Quercetin decreased cell viability level in a dose-dependent manner to almost zero at 100 μm. Up to this concentration, it did not induce significant apoptosis nor did it decrease the survival-fractions below 90% during a 4 d follow-up. The data suggest that Quercetin is lethal to melanoma cells at concentrations that do not activate apoptosis during the first 4 d post-exposure and that quercetin’s effects extend beyond the period of direct contact. Both quercetin and UVB induced late-type apoptosis at the upper range of the tested doses, but they do not appear to share all the pathways that they activate. Finally, this paper provides novel data showing that quercetin causes two different lethal effects on human melanoma cells, suggesting the activation of at least two different dose-depended mechanisms.

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Various extracts of aerial parts of Varthemia (Varthemia iphionoides Boiss) were investigated for radical-scavenging activity, antioxidative activity, and porcine pancreas α-amylase inhibitory activity. The ethanol and water extracts showed a pronounced 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, with inhibition of about 90% at a concentration of 100 μg/ml, and α-amylase inhibitory activity of about 70% at a concentration of 200 μg/ml by the 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G₃) degradation method. The ethanol extract was purified by column chromatography to give seven 3-methoxyflavones (1–7) and eudesmane sesquiterpene, selina-4,11(13)-dien-3-on-12-oic acid (8). The structures of these compounds were established by NMR, MS, and UV spectroscopy. Of 3-methoxyflavones, 5,7,4′-trihydroxy-3,6-dimethoxyflavone (1), 5,7,4′-trihydroxy-3,3′-dimethoxyflavone (2), and 5,4′-dihydroxy-3,7,3′-trimethoxyflavone (3,7,3′-tri-O-methyl-quercetin) (7) exhibited pronounced radical-scavenging activity. The antioxidative activity in the linoleic acid system was considerable in compounds 1, 2, and 5,4′-dihydroxy-3,6,7-trimethoxyflavone (4). Compounds 1, 2, 4, 5 (5,7,4′-trihydroxy-3-methoxyflavone), and 6 (5,4′-dihydroxy-3,7-dimethoxyflavone) showed markedly high inhibitory activity against porcine pancreas α-amylase. Eudesmane sesquiterpene did not show any activity.

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Using the shuttle vector pBU4, the mosquitocidal toxin gene mtx1 from Bacillus sphaericus strain SSII-1 was introduced into an acrystalliferous strain of B. thuringiensis both individually and in combination with the accessory protein gene p20 and the cytolytic protein gene cyt1Aa from B. thuringiensis subsp. israelensis. Bioassay results indicated that the recombinants B-pMT4(Mtx1) and B-pMT9(Mtx1), both individually containing mtx1, had moderate toxicities to binary toxin susceptible and binary toxin resistant Culex quinquefasciatus larvae during the vegetative growth stage, but that their toxicities declined rapidly during the sporulation phase. The LC₅₀ values were 2.5 and 4.8 mg/ml respectively, against 3–4 instar susceptible and resistant larvae for the final sporulated cultures of recombinants B-pMT9(Mtx1), and little toxicity was detected for B-pMT4(Mtx1). Meanwhile, the recombinant B-pMPX2(Mtx1+Cyt1Aa) expressing Mtx1, P20 alone, and Cyt1Aa in combination had stable toxicities during both the vegetative phase and the sporulation phase, with a LC₅₀ ranging from 0.45–0.58 mg/ml. Furthermore, expression of Cyt1Aa appeared to enhance the activity of Mtx1 to target mosquito larvae, suggesting a synergism between Cyt1Aa and Mtx1 toxins.

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EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile.

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Escherichia coli AP endonuclease (ExoIII) and its human homolog (APE1) have the sole tryptophan residue for AP site recognition (AP site recognizer) but these residues are at different positions near the catalytic sites. On the other hand, many bacterial AP endonucleases have two tryptophan residues at the same positions of both ExoIII and APE1. To elucidate whether these residues are involved in AP site recognition, the ExoIII homologs of Thermoplasma volcanium and Lactobacillus plantarum were characterized. These proteins showed AP endonuclease and 3′-5′exonculease activities. In each enzyme, the mutations of the tryptophan residues corresponding to Trp-280 of APE1 caused more significant reductions in activities and binding abilities to the oligonucleotide containing an AP site (AP-DNA) than those corresponding to Trp-212 of ExoIII. These results suggest that the tryptophan residue corresponding to Trp-280 of APE1 is the predominant AP site recognizer, and that corresponding to Trp-212 of ExoIII is the auxiliary recognizer.

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Cycad leaf lectin (CRLL), a mannose-recognizing jacalin-related lectin (mJRL), was first cloned as a gymnosperm lectin and expressed. The cDNA sequence of CRLL (DDBJ, accession no. AB198328), coding 291 amino acid residues, has a tandem repeat of about 150 amino acids divided into N- and C-terminal domains as Japanese chestnut mJRL. Sequence alignment showed deletion and insertion of the sequence, and its putative carbohydrate-binding sites showed some differences from other JRLs. PCR analysis showed that this lectin was expressed in the cycad leaf but not in the root or seed. Recombinant CRLL (rCRLL) was expressed in Escherichia coli and purified by affinity chromatography after refolding procedures. Properties of active rCRLL appeared to be almost the same as those of native CRLL.

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Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k_cat⁄K_m^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.

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Carotovoricin Er (CtvEr), which is produced by a plant soft rot disease causative agent, Erwinia carotovora subsp. carotovora Er, is a high-molecular-weight bacteriocin showing Myoviridae phage-tail-like morphology with contractile sheath and plural tail fibers. We determined the complete nucleotide sequences of CtvEr genes on the E. carotovora Er chromosome and report that CtvEr genes consist of lysis cassette, major and minor structural protein gene clusters. Four promoters were identified. The lysis gene cassette, which is composed of the genes for lysis enzyme and holin, was also identified and characterized. The nucleotide sequences and organization of the genes for CtvCGE, which is produced by E. carotovora strain CGE234-M403 with the morphology similar to CtvEr, were also determined and compared to that of CtvEr, and it was found that CtvCGE is almost identical to CtvEr except for tail fibers which are involved in the killing spectra of both bacteriocins. We also explain that the gene organization and the deduced amino acid sequences of both carotovoricins are very close to those of prophage, which is lysogenized in the chromosome on Salmonella enterica serovar Typhi CT18. These findings strongly suggest that Ctv evolved as a phage tail-like bacteriocin from a common ancestor with Salmonella typhi prophage.

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Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.

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A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.

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RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

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A series of chemical analogs of sodium houttuynin, the major constituent in the volatile oil of the perennial plant Houttuynia cordata Thunb, were studied for in vitro inhibition of renin activity. At a reaction concentration of 0.196 mg/ml, each of the sodium houttuynin analogs (SHAs), viz., hexanoyl acetal sodium sulfite (SHA-C6), octanoyl acetal sodium sulfite (SHA-C8), decanoyl acetal sodium sulfite (SHA-C10), dodecanoyl acetal sodium sulfite (SHA-C12) and tetradecanoyl acetal sodium sulfite (SHA-C14), inhibited renin activity up to 34.37%, 44.03%, 79.33%, 83.04%, and 93.19% respectively. The IC₅₀ values (the concentration of SHA that is required to reduce renin activity by 50%) of the most potent analogs were 273, 195, and 44 μM for SHA-C10, SHA-C12, and SHA-C14 respectively. Kinetic studies with SHA-14 indicated a linear mixed-type of enzyme inhibition with a K_i value of 45.35 μM. It was concluded that the SHAs constitute potentially useful ingredients for the formulation of antihypertensive products.

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UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.

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Glutamine synthetase (GS) is a key enzyme for nitrogen assimilation. Although GS contains multiple molecular species found in plastid, mitochondria and cytoplasm in green plants and algae, genome analysis of a red alga, Cyanidioschyzon merolae, revealed a single nuclear gene for GS (CmGS). In this study, we experimentally determined the CmGS localization in the cytoplasmic compartment.

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The vanillate demethylase genes from Streptomyces sp. NL15-2K were cloned and sequenced. The vanA and vanB gene homologs, which encode the terminal oxygenase subunit (VanA) and the ferredoxin-type reductase subunit (VanB) of the enzyme respectively, were found in the sequenced 7.5-kb DNA region. Expression of the vanAB genes in Streptomyces lividans 1326 resulted in in vivo demethylation of veratric acid to vanillic acid.

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LitR is a MerR-family regulator that controls light-induced carotenogenesis in Streptomyces coelicolor. The introduction of litR conferred photo-dependent transcription activities on Streptomyces griseus, which does not perform photo-responsive carotenogenesis. LitR discovers homologs encoded at illumination-related loci of various non-phototrophic bacterial genomes. Probably, LitR acts not only as a transcriptional regulator but also a photosensor.

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Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5′ untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5′ UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the β-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream β-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

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We investigated the anti-tumor effects of a dry powder preparation of the antlered form of Ganoderma lucidum (G. lucidum AF, rokkaku-reishi in Japanese), a variant type of G. lucidum, not only in allogeneic Sarcoma 180-bearing ddY mice, but also in syngeneic MM 46-bearing C3H/He mice. G. lucidum AF inhibited tumor growth and elongated the life span when orally administered to mice by free-feeding of a 2.5% G. lucidum AF-containing diet. It also showed anti-tumor activity in spite of post-feeding after tumor inoculation. G. lucidum AF significantly countered the depression of splenic CD8⁺ cells and protected the decrease in interferon-gamma (IFN-γ) production in regional lymph nodes of MM 46-bearing mice, indicating that the anti-tumor activity of G. lucidum AF might be caused by its immunostimulating action. These results suggest that the ingestion of G. lucidum AF can be useful for the prevention and curing of cancer.

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Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5–1 mol of dextran could be bound to 1 mol of native BSA. SDS–PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95–2.35×10⁵ g/mol and a less-abundant fraction with aggregates greater than 1.50×10⁶ g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.

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The effects of supplemental D-psicose in the diet on diurnal variation in plasma glucose and insulin concentrations were investigated in rats. Forty-eight male Wistar rats were divided into four groups. Each group except for the control group was fed a diet of 5% D-fructose, D-psicose, or psico-rare sugar (3:1 mixture of D-fructose and D-psicose) for 8 weeks. Plasma glucose levels were lower and plasma insulin levels were higher at all times of day in the psicose and psico-rare sugar groups than in the control and fructose groups. Weight gain was significantly lower in the psicose group than in the control and fructose groups. Liver glycogen content, both before and after meals was higher in the psicose group than in the control and fructose groups. These results suggest that supplemental D-psicose can lower plasma glucose levels and reduce body fat accumulation. Hence, D-psicose might be useful in preventing postprandial hyperglycemia in diabetic patients.

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To compare the systemic efficacy of borage oil (Borago officinalis: BO) and gromwell (Lithospermum erythrorhizon), two plant species of the Boraginaceae family, epidermal hyperproliferation was induced in guinea pigs by a hydrogenated coconut oil diet for 8 weeks. Subsequently, guinea pigs were fed diets of BO (group HBO), organic extract (group HGO), or water extract (group HGW) of gromwell for 2 weeks. In groups HGO and HGW, proliferation scores and the level of ceramides, the major lipid maintaining epidermal barrier, were similar with those in normal control group BO fed BO diet for 10 weeks. Despite accumulation of 15-hydroxyeicosatrienoic acid (15-HETrE), the potent anti-proliferative metabolite of γ-linolenic acid (GLA: major polyunsaturated fatty acid in BO), the reversal of epidermal hyperproliferation and the ceramide level of group HBO were less than those of groups HGO and HGW. Taken together, our data demonstrate that gromwell is more effective in reversing epidermal hyperproliferation with a marked increase in ceramides.

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A soybean protein isolate (SPI), and its β-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS–PAGE of the β-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 °C showed that the amount of the monolayer adsorption of these preparations was about 6–9%, the value for the β-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The β-conglycinin and glycinin preparations were respectively denatured at around 75 °C and 86 °C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.

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We aimed to clarify whether grape seed polyphenols (GSPs) are candidates therapeutic agents against diabetes mellitus, and to determine what degree of GSP oligomerization has the most potent efficacy. We studied the protective effects of various molecular weight GSPs (monomer, oligomer, polymer, and oligonol) on high glucose-induced cytotoxicity. In the present study, a high concentration of glucose (30 mM) induced cytotoxicity and oxidative stress (reactive oxygen species and nitric oxide) in cultured LLC-PK₁ cells, but treatment with GSPs, especially oligomer GSPs, had potent protective effects against high glucose-induced oxidative stress. In addition, high glucose induced nuclear translocation of nuclear factor-kappa B, and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and bax, but GSP treatment inhibited them. These results indicate that GSPs have protective effects against high glucose-induced cytotoxicity, and among them, oligomer GSPs have more potent effects than other GSPs (monomer, polymer, and oligonol) on high glucose-induced renal cell damage.

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The activities of β-carotene on redox status and the immune functions of RAW264 cells, a murine macrophage cell line, were investigated. Supplementation with β-carotene for RAW264 cells resulted in apparently inconsistent redox indices: lipid peroxidation was enhanced but intracellular oxidation was moderately attenuated. Attenuated intracellular oxidation was endorsed by an increase in glutathione accompanied by up-regulated transcription of a subunit of γ-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis. α-Tocopherol, which can quench lipid peroxidation by free radical, neither inhibited that by β-carotene nor influenced the intracellular redox status. Lipopolysaccharide-stimulated transcriptions of IL-1β and IL-12 p40 in RAW264 were inhibited by β-carotene but not by α-tocopherol. These results indicate that β-carotene, which can modulate the intracellular redox status of macrophages by enhancing the level of intracellular glutathione, is related to the immune functions of macrophages.

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We compared the dietary effects of dihomo-γ-linolenic acid (DGLA) contained in the DGLA oil produced by a fungus with γ-linolenic acid (GLA) on the fatty acid composition. Wistar rats were fed with three kinds of oil for two weeks as follows: (i) control group: corn oil; (ii) GLA group: borage oil; (iii) DGLA group: DGLA oil/safflower oil = 55:45. The DGLA concentrations in the liver, serum, and brain of the DGLA group were higher than those of the GLA oil group. We also examined the dose effect of DGLA. The DGLA levels in the liver, serum, and brain significantly increased with increasing dosage of DGLA in the diet. DGLA administration significantly increased the ratio of PGE₁/PGE₂ in the rat plasma. The mechanism for GLA administration to improve atopic eczema is thought to involve an increase in the concentration of DGLA metabolized from GLA, so these results suggest that the dietary effect of DGLA would be more dominant than GLA.

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Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.

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Oxidative stress is closely associated with diabetes and is a major cause of insulin resistance. Impairment of hepatic insulin action is thought to be responsible for perturbations in hepatic glucose metabolism. In this study, we found that oxidative stress is involved in the dysregulation of gene expression of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, by a mechanism independent of insulin. Elevation of oxidative stress by injection of ferric nitrilotriacetate in rats increased the expression of hepatic PEPCK mRNA. To examine the direct action of oxidative stress on PEPCK expression, we treated H4IIE hepatoma cells with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO increased intracellular oxidative stress and the expression of PEPCK mRNA. Inhibition of p38 mitogen-activated protein kinase (p38 MAP kinase), which mediates responses to oxidative stress, suppressed the induction of PEPCK mRNA by BSO. These results suggest that oxidative stress dysregulates hepatic PEPCK expression by an insulin-independent mechanism.

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The protective effects of red potato extract (RPE) as to liver damage were determined in <small>D</small>-galactosamine (GalN)-intoxicated rats. Increases in serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activities, all of which were induced by GalN injection, decreased in RPE administered rats, suggesting that RPE acts as a functional food showing anti-hepatotoxicity.

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In this study, we found that antihypertensive di-peptide Val-Tyr (VY) showed a vascular relaxation effect in KCl-induced contraction of thoracic aorta rings from 18-week-old spontaneously hypertensive rats among di-peptides of VY, Ile-Tyr, and Tyr-Val irrespective of their angiotensin I-converting enzyme inhibitory activity. The effect was endothelium-independent, and was closely associated with vascular responses in the vascular smooth muscle layer.

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RNA interference (RNAi) has been widely used for the analysis of gene function and represents a new promising approach to develop effective antiviral drugs. In this study, several small interfering RNAs (siRNAs) corresponding to two structural genes (core and E2) of hepatitis C virus (HCV) were designed and in vitro transcribed to explore the possibility of silencing these two genes. The plasmids pEGFP-C and pEGFP-E2, which contain the EGFP reporter gene and the core or E2 gene as silencing targets, were co-transfected with siRNAs into HEK 293T cells. At various time points of post-transfection, core and E2 expression levels were detected by fluorescence microscopy, flow cytometry, Western blotting, and real-time quantitative PCR. The results showed that the mean fluorescence intensity, protein expression, and RNA transcripts of siRNAs transfected cells were significantly reduced. This may provide an approach for the development of novel prophylactic or therapeutic agents for HCV infection.

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A semi-continuous four-channel colon simulator was used to study the effects of lactose for the first time on the growth and fermentation dynamics of colonic microbiota. In six separate simulations, lactose supplementation increased the total SCFA concentration by 3–5 fold as compared with the baseline in the respective vessels. The total bacterial density was inversely correlated with lactic acid production (P=0.003), while production of butyrate (P=0.007) and propionate (P=0.02) correlated with higher numbers of bacteria. A major shift in the microbial community structure in the lactose supplemented vessels was demonstrated by bacterial genomic %G+C-profiling of the total population, where lactose supplementation induced a clearly dominant peak in the bifidobacteria prominent area, %G+C 60–65. The transient shift to increased numbers of bifidobacteria (23–27%) of all bacteria in the first two vessels was also confirmed by the bifidobacteria-specific QPCR-method. In conclusion, lactose produced dramatic changes in microbiota composition and activity as compared with the baseline fermentation.

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Two novel genes (tsB, tsC) involved in the conversion of DL-2-amino-Δ²-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine through S-carbamyl-L-cysteine (L-SCC) pathway were cloned from the genomic DNA library of Pseudomonas sp. TS1138. The recombinant proteins of these two genes were expressed in Escherichia coli BL21, and their enzymatic activity assays were performed in vitro. It was found that the tsB gene encoded an L-ATC hydrolase, which catalyzed the conversion of L-ATC to L-SCC, while the tsC gene encoded an L-SCC amidohydrolase, which showed the catalytic ability to convert L-SCC to L-cysteine. These results suggest that tsB and tsC play important roles in the L-SCC pathway and L-cysteine biosynthesis in Pseudomonas sp. TS1138, and that they have potential applications in the industrial production of L-cysteine.

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The structure of the hydrolyzed product (F-2) with a molecular mass of about 2 kDa released from γ-polyglutamic acid by the γ-glutamyl hydrolase YwtD of Bacillus subtilis was analyzed. The results showed that F-2 is an optically heterogeneous polymer consisting of D- and L-glutamic acid in an 80:20 ratio with D-glutamic acid on both the N- and C-terminal sides, suggesting that YwtD is an enzyme that cleaves the γ-glutamyl bond between D- and D-glutamic acid recognizing adjacent L-glutamic acid toward the N-terminal region.

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L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine α-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.

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A gene for cytochrome P450 (moxA) from Nonomuraea recticatena, coexpressed with camAB for pseudomonad redox partners in Escherichia coli, hydroxylated oleanolic acid to produce queretaroic acid. When we used the P450-induced whole-cell as a catalyst, only a small amount of queretaroic acid was produced, probably due to poor permeability of oleanolic acid into the E. coli cell. In an alternative approach with the cell-free reaction system, the conversion ratio increased up to 17%.

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