We synthesized chondroitin and its sulfate E hexasaccharides (1 and 2) composed of the trimer of the repeating disaccharide, β-D-GalNAc(±4,6-di-O-SO₃Na)-(1→4)-β-D-GlcA, by employing an efficient synthetic strategy for longer chondroitin oligosaccharide. Successful elongation with the β-D-GalNAc-(1→4)-β-D-GlcA unit instead of the corresponding disaccharide possessing an azide group avoided problematic reduction of the multiple azide groups on the hexasaccharide.

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A structurally unique glucosinolate (GSL) was identified to be 4-(β-D-glucopyranosyldisulfanyl)butyl GSL in rocket leaves. The positive-ion electrospray ionization mass spectrometry (ESI-MS) data indicated that the new GSL had a molecular weight of 521 (m⁄z 522, [M+H]⁺, as desulfo-GSL). The molecular formula of the substance was determined to be C₁₇H₃₂O₁₁NS₃ (m⁄z 522.1143, [M+H]⁺) based on its positive-ion high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) data. For the further confirmation, desulfated GSL of 4-(β-D-glucopyranosyldisulfanyl)butyl GSL was prepared by commercial 1-thio-β-D-glucose and dimeric 4-mercaptobutyl desulfo-GSL, which was also isolated from rocket leaves, and its chemical structure was then confirmed by MS data and nuclear magnetic resonance (NMR) spectroscopy. In addition, the antioxidative activity of 4-(β-D-glucopyranosyldisulfanyl)butyl desulfo-GSL was measured by means of chemiluminescence (CL) for evaluating the functional properties. The antioxidative activity (2.089 unit/g) was relatively higher than that of dimeric 4-mercaptobutyl desulfo-GSL (1.227).

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Evaluating the binding energy of a catechin/caffeine complex in water is important in order to elucidate the ability for molecular recognition of tea catechins. The results of this study revealed that the stoichiometric ratio of the complexation between tea chatechins (EGCg, ECg, EGC, and EC) and caffeine was 1:1 at least up to a concentration of 5.0 mM. The free energy (−ΔG) values for binding in water at 301 K were evaluated to be 2.7, 2.6, 2.2, and 2.0 kcal/mol for EGCg, ECg, EGC, and EC, respectively, by the titration method with ¹H-NMR. An investigation of the ¹H-NMR chemical shift change and NOESY spectra in the catechin/caffeine solutions showed the participation of the A-rings of the catechins in complexation, as well as that of the galloyl groups or B-rings.

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We examined the biosynthetic pathway of abscisic acid (ABA) after isopentenyl diphosphate in a fungus, Cercospora cruenta. All oxygen atoms at C-1, -1, -1′, and -4′ of ABA produced by this fungus were labeled with ¹⁸O from ¹⁸O₂. The fungus did not produce the 9Z-carotenoid possessing γ-ring that is likely a precursor for the carotenoid pathway, but produced new sesquiterpenoids, 2E,4E-γ-ionylideneethane and 2Z,4E-γ-ionylideneethane, along with 2E,4E,6E-allofarnesene. The fungus converted these sesquiterpenoids labeled with ¹³C to ABA, and the incorporation ratio of 2Z,4E-γ-ionylideneethane was higher than that of 2E,4E-γ-ionylideneethane. From these results, we concluded that C. cruenta biosynthesized ABA by the direct pathway via oxidation of ionylideneethane with molecular oxygen following cyclization of allofarnesene. This direct pathway via ionylideneethane in the fungus is consistent with that in Botrytis cinerea, except for the positions of double bonds in the rings of biosynthetic intermediates, suggesting that the pathway is common among ABA-producing fungi.

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Novel acremolactones B and C were isolated from the acremolactone A-producing Acremonium roseum I4267. The structure of acremolactone B having a phenylpyridyl γ-lactone was elucidated by spectroscopic methods. It showed plant growth inhibitory activity toward Chinese cabbage seedlings. The congener of acremolactone C having a phenylcyclopentenone γ-lactone showed weak activity.

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The Solanum abutiloides plant is highly resistant to soil-borne pathogens such as Fusarium oxysporum f. sp. melongenae, Verticillium dahliae, and Ralstonia solanacearum. This species is utilized as a mating source of resistant cultivars and is also used as a rootstock. The root exudate of Solanum abutiloides was extracted from a soil system composed of charcoal and vermiculite. Anti-fungal activity was found in the extract, and an active ingredient was isolated. The chemical structure of the active compound was determined to be 3-β-acetoxysolavetivone, a new sesquiterpenoid. The anti-fungal activity of 3-β-acetoxysolavetivone examined by the inhibition of spore germination of Fusarium oxysporum was close to that of lubimin, and higher than that of solavetivone.

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The dihydroxy C₅₀-carotenoid, decaprenoxanthin, was produced by Aureobacterium sp. collected from sea water. The addition of D-psicose to the culture medium improved the growth of cells and the yield of the carotenoid. The ¹³C-NMR spectrum of decaprenoxanthin, which has not previously been reported, was successfully measured.

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Thermally decomposed products of (±)-linalyl β-D-glucoside were analyzed by GC and GC/MS. 2,6-Dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl β-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.

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The NADP⁺-preferring glucose dehydrogenase from thermoacidophilic archaeon Thermoplasma acidophilum has been characterized, and its crystal structure has been determined (Structure, 2:385-393, 1994). Its sequence and structure are not homologous to bacterial NAD(P)⁺-dependent glucose dehydrogenases, and its molecular weight is also quite defferent. On the other hand, three functionally unknown genes with homologies to bacterial NAD(P)⁺-dependent glucose dehydrogenases have been sequenced as part of the T. acidophilum genome project (gene names: Ta0191, Ta0747, and Ta0754 respectively). We expressed two genes of three, Ta0191 and Ta0754, in Escherichia coli, and purified the gene products to homogeneity. Dehydrogenase activities were thereby detected from the purified proteins. The Ta0754 gene product exhibited aldohexose dehydrogenase activity, and the Ta0191 gene product exhibited weak 2-deoxyglucose dehydrogenase activity. No aldohexose dehydrogenase gene has been isolated, while the enzyme was reported in 1968. This is the first report of the gene and primary structure. The purified Ta0754 gene product, designated AldT, was characterized. The enzyme AldT effectively catalyzed the oxidation of various aldohexoses, especially D-mannose. Lower activities on D-2-deoxyglucose, D-xylose, D-glucose, and D-fucose were detected although no activities were shown on other aldohexoses or additional sugars. As a cofactor, NAD⁺ was much more suitable for the activity than NADP⁺. The NAD⁺-preferring dehydrogenase most effectively reacting to D-mannose is for the first time. AldT was most active at pH 10 and above 70 °C, and completely stable up to 60 °C after incubation for 15 min. Other enzymatic properties were also investigated.

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We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 °C for the original form, of 74 °C for the dephosphorylated form, and of 71 °C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 °C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 °C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.

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The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 °C, but also at 10 °C, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 °C and 12 °C respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

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The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined. The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO. Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO. Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells. Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol. The transcription of three genes, encoding an aa₃-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction. The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells. A model is proposed for the oxidation of reduced inorganic sulfur compounds in A. ferrooxidans NASF-1 cells.

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A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-α-glucosyltransferase (6GT) and 3-α-isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.

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An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS–PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala–Phe–Met–Lys–Tyr–X–Thr–Thr–Gly–Gly–Pro–Val–Ala–Gly–Lys–Ile–Asn–Val–His–Leu–. The α-mannosidase activity for Man₅GlcNAc₁ was enhanced by the addition of Co²⁺, but the addition of Zn²⁺, Ca²⁺, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man₆GlcNAc₁ was significantly faster than that for pyridylaminated Man₆GlcNAc₂, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013–1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co²⁺ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.

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The 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example for mitochondrial HSP70, which exists in a stable complex with a nucleolytic subunit as a multi-site specific DNase. The HSP70-subunit in Endo.SceI was autophosphorylated by ATP in vitro. The autophosphorylation was higher in the Endo.SceI complex form than in the free form. Although the autophosphorylation had no significant effect on the endonucleolytic activity of Endo.SceI, the factors favoring autophosphorylation protected the endonucleolytic activity of Endo.SceI against heat inactivation. ATP, adenosine 5′-O-(3-thiotriphosphate) (ATP-γ-S), and ADP not only protected the endonucleolytic activity against heat inactivation in the presence of Ca²⁺ ions, but also reduced the labeling of the HSP70-subunit by [γ-³²P]ATP in Endo.SceI. These findings suggest that the HSP70-subunit shields Endo.SceI from heat inactivation through ATP/ADP binding.

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Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc₂ (3.4%), Man₂FucGlcNAc₂ (2.1%), Man₃FucGlcNAc₂ (2.5%), Man₃FucXylGlcNAc₂ (42.5%), two isomers of Man₂FucXylGlcNAc₂ (39.1%), Man₃XylGlcNAc₂ (6.5%), and Man₂XylGlcNAc₂ (3.9%).

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We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)–receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level. To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits. Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR–RLKs. This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction. Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.

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We cloned and sequenced a recQ gene homolog from the basidiomycetous mushroom Lentinula edodes (Le.). This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa) and be interrupted by 11 small introns. The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation on a sawdust-corn bran medium. The L. edodes dikaryotic mycelial cells, which had been vegetatively grown in SMY liquid medium, were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homolog S. cerevisiae SGS1 gene.

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Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown. A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI). IRKI is a novel but highly conserved protein found in higher plants. The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems. In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells. IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.

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Secondary wall thickenings in tracheary elements were specifically stained by incubation of Arabidopsis and maize in Silver Stain Plus (Bio-Rad) staining solution, after pretreatment with SDS and ethanol solution. Scanning electron microscopic analysis of sections of celery revealed that silver particles were deposited on the secondary wall thickenings, indicating that the staining was due to the deposition of silver through the interaction of the stain with lignin. This method is more sensitive than the acidified phloroglucinol method.

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We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A⁶²G⁶³G⁶⁴A⁶⁵) was replaced with GGA (denoted ΔA), GA (ΔAG), A (ΔAGG), AAGGA (ΣA), AAAGGA (ΣAA), and AAAAGGA (ΣAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>ΣAA>ΣA>ΣAAA>>ΔAG>ΔA, ΔAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.

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We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 μM to 10 μM.

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Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K_i of 6.1–19.4% (v/v) or 0.66–4.80 M. From the relationship between the structures of alcohols and their K_i values, alcohols are considered to bind the hydrophobic S1′ subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.

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A hot water extract obtained by boiling adzuki beans (Vigna angularis) to produce bean paste for Japanese cake showed inhibitory activity against alpha-glucosidase, alpha-amylase, maltase, sucrase, and isomaltase after HP-20 column chromatography. The IC₅₀ values for each hydrolylase were 0.78 mg/ml (α-amylase), 2.45 mg/ml (maltase), 5.37 mg/ml (sucrase), and 1.75 mg/ml (isomaltase). The active fraction showed potential hypoglycemic activity in both normal mice and streptozotocin (STZ)-induced diabetic rats after an oral administration of sucrose, but did not show any effect on the blood glucose concentration after glucose administration, suggesting that the active fraction suppressed the postprandial blood glucose level by inhibiting α-glucosidase and α-amylase, irrespective of the endogenous blood insulin level.

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Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of “Koganesengan”, the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.

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This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)₂D₃ concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6–0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)₂D₃ concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)₂D₃.

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Human sera obtained from children with egg allergy reacted well with both native and heated ovomucoid (OM). Ovalbumin is present in egg white in a 5 times greater quantity than OM; however, it easily aggregates and becomes difficult to extract by heating. For accurate food allergen labeling of processed food, therefore, OM should be evaluated with the determination of egg white protein in consideration of heat denaturation. Three kinds of monoclonal antibodies and sandwich ELISA tests were established which are able to recognize the native and/or heat-denatured forms of OM. The usefulness of these characteristic mAbs and ELISA tests are discussed in relation to allergen labeling, monitoring food processing, and movement or change of dietary protein in vivo.

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We previously examined wogonin, isolated from Scutellaria baicalensis, chemical mediators, and IgE by mesenteric lymph node (MLN) lymphocytes in rats. The present study explores the effect of wogonin on the MLN lymphocyte function of mice given orally at 20 mg/kg for 2 weeks with dextran sulfate sodium (DS)-induced colitis. The results indicate that IgA levels in MLN lymphocytes were high, while IgE was low, in mice given wogonin compared to those given water. Also, fecal IgA concentration of DS in the wogonin group mice was significantly higher than in the DS group. Concentrations of interferon-γ and interleukin (IL)-2 of T cells by concanavalin A treatment was significantly higher in the wogonin fed group than in the normal group. Activation-induced IL-4, IL-5 and IL-10 secretion was lower in wogonin fed mice compared control mice after DS-induced colitis. For these reasons, we conclude that wogonin can alleviate the inflammation in DS-induced colitis brought about by an abnormal Th₂ response.

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The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 μM digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.

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Seven-week old female rats fed restricted foods including the fish oils Docosahesaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) and perilla oil with food intake decreased by 50%, had increases of fracture force and bone mineral density (BMD) and decreases in levels of Deoxypiridinoline (Dpd) and Calcium (Ca) in the urine, compared with those of rats with osteoporosis due to restricted soy bean oil food intake. Therefore, the fish oils DHA and EPA and perilla oil depressed excretion of urinary Ca and inhibited osteoporosis due to restricted food intake.

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Five phenolic compounds, p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, and iso-americanol A, were found first time in the barley tea, together with the known compounds, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and p-coumaric acid. The anti-oxidative properties were evaluated by measuring their peroxynitrite-scavenging activities. Among these compounds, 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, and isoamericanol A showed stronger activities than that of BHT (butylated hydroxytoluene) at 400 μM.

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Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as γ-oryzanol.

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A determination was made of fatty acid compositions in twelve commercial red wines made from grapes differing in kind and vintage. Twelve fatty acids were identified, palmitic, myristic, and lauric acids being found predominant. Total acyls (32∼81 nmol/100 ml) differed considerably. Changes in fatty acid constituents in must from grape berries and wines according to the process of manufacture were also examined.

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The physiological effects of 9cis,11trans,13cis-conjugated linolenic acid (9c,11t,13c-CLNA), one of the CLNA isomers, were studied in human hepatoma HepG2 cells. 9c,11t,13c-CLNA significantly decreased apolipoprotein B100 secretion compared with α-linolenic acid (α-LNA). The uptake of ¹⁴C-oleate into newly synthesized cellular triacylglycerol was also decreased by 9c,11t,13c-CLNA more than by α-LNA treatment. This is the first study to show the hypolipidemic effect of 9c,11t,13c-CLNA.

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We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine κ-casein. The amino acid sequence of this peptide is Phe–Leu–Pro–Tyr–Pro–Tyr (FLPYPY), corresponding to peptidic sequence 76–81 of bovine κ-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10⁻⁹ M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76–80 and 76–82 of bovine κ-casein respectively, were ineffective.

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“Isolation of Lactobacillus sakei Strain KJ-2008 and Its Removal of Characteristic Malodorous Gases under Anaerobic Culture Conditions” by Jeong-Dong KIM and Kook-Hee KANG, Bioscience, Biotechnology, and Biochemistry , Vol.68, No.12, pp. 2427-2435, December 2004.
This manuscript was canceled on October 23, 2013 because it was found to be a duplicate submission.

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Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.

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To broaden our understanding of extracellular proteins of Aspergillus oryzae at the conidial germination stage, analyses of the secreted proteins during germination were carried out. Taka-amylase A (TAA), glucoamylase (GLAA), and aspergillopepsin A (PEPA) were identified as the main products by peptide mass fingerprinting. TAA and PEPA were detected simultaneously with the formation of germ tubes. With the development of germination, the pH of the medium fell from 5.5 to 3.5. The secreted PEPA had a pro-sequence and likely shifted from 42 kDa to 41 kDa below pH 4.6, indicating that the precursor of PEPA was secreted and underwent pH-dependent processing. Furthermore, the 41 kDa protein was trapped by the addition of pepstatin A, the specific inhibitor of PEPA, suggesting that the maturation of pro-PEPA was a stepwise autoprocessing upon acidification of the medium and itself was an intermediate of the processing. It was implied that PEPA plays an important role at the early germination stage.

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