(Vol.63 No.8 1999)
-1-
Effects of High Pressure on Lipids and Biomembranes for Understanding
High-Pressure-Induced Biological Phenomena
Michiko KATO and Rikimaru HAYASHI・
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, JapanThis review covers high pressure effects on lipids, lipid bilayers, and biochemical observations recently found in the field of high-pressure bioscience and biotechnology including deep-sea microbiology and food science. To explain these phenomena in a unified model, recent studies of physical and chemical properties of artificial membranes and natural membranes are summarized. On the basis of this newly described knowledge, high pressure effects on biochemical events are considered at the molecular level and concluded that high pressure induces decreases in biomembrane fluidity and phase transitions that result in breakage of the membrane, and finally, leads to the destruction of bilayer membrane accompanied by denaturation of membrane-associated proteins.
high pressure; biomembrane; pressure-induced membrane destruction; phospholipid bilayer
-2-
Characterization of Chimeric Enzymes Constructed between Two Distinct
α-Amylase cDNAs from Cultured Rice Cells
Rei ABE,1 Kensuke YOSHIDA,1 Masanobu AOYAGI,1 Shin KASAHARA,1 Eiji ICHISHIMA,2
and Tasuku NAKAJIMA1,・1Laboratory of Enzymology, Division of Life Science, Graduate School of Agricultural Science,
Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai-shi 981-8555, Japan
2Department of Bioengineering, Faculty of engineering, Soka University, 1-26 Tankicho, Hachioji,
Tokyo 192-8577, JapanReceived November 12, 1998; Accepted April 14, 1999
Cultured cells of rice (Oryza sativa cv Sasanishiki) produce two α-amylase isozymes, AMY-I and AMY-III. Using a bacterial expression system, eight chimeric genes constructed with various combination of AMY-I and AMY-III cDNA fragments were expressed, and each recombinant chimeric protein was characterized. Four of the eight recombinant enzymes having region c (one of the four regions having unconserved base sequences between AMY-I and AMY-III cDNAs) of AMY-I showed the same enzyme characteristics as that of native AMY-I, which had high temperature optimum at 50°C. The other four chimeric proteins carrying region c of AMY-III showed the AMY-III type characteristics, which were a low temperature optimum at 25°C and susceptibility to a higher maltooligosaccharide (G17) substrate. The unconserved region c is involved in the decision of the characteristic of AMY-I or AMY-III.
α-amylase; gene expression; chimeric protein; suspension-cultured rice cells
-3-
Kinetic Analysis of Temperature-Programmed Autoxidation
of Ethyl Eicosapentaenoate and Ethyl Docosahexaenoate
Hidefumi YOSHII, Takeshi FURUTA, Takatoki ITOH, Yoshihisa MISAWA,＊
Noriaki HATA,＊ Hideaki KOBAYASHI,＊＊ Yu-Yen LINKO,＊＊＊ and Pekka LINKO＊＊＊Department of Biotechnology, Tottori University Tottori 680-0945, Japan
＊Harima Chemicals, Inc., Tsukuba 300-2635, Japan
＊＊R＆D Div., Q.P. Corporation, Tokyo 183-0034, Japan
＊＊＊Department of Chemical Technology, Helsinki University of Technology,
Espoo, FIN-02015 HUT, FinlandReceived December 8, 1998; Accepted May 6, 1999
On the basis of an autocatalytic and a first order reaction||kinetics, a nonisothermal oxidation reaction model was||developed for n-3 polyunsaturated fatty acid (PUFA) under temperature-programmed linear heating conditions. With this model, the activation energy of oxidative reaction can easily be obtained with at least three linear heating conditions. The temperature-programmed oxidation experiments of ethyl eicosapentaenoate and ethyl docosahexaenoate were done under linear heating conditions of 4 to 12 K/h. The activation energies and the frequency factors obtained were in good agreement with those by the isothermal oxidation experiments reported previously.
||temperature-programmed autoxidation; DHA; ||EPA; kinetic energy
-4-
Physiological Effects of Water-soluble Soybean Fiber in Rats
Taro TAKAHASHI,・ Hirokazu MAEDA,＊ Toshiaki AOYAMA,＊＊ Takashi YAMAMOTO,＊＊
and Kiyoharu TAKAMATSU＊＊Novelty Materials Research Institute, Tsukuba R＆D Center, Fuji Oil Co. Ltd., 4-3 Kinunodai,
Yawara, Tsukuba-gun, Ibaraki 300-2497, Japan
＊Protein Development Dept. II, Hannan R＆D Center, Fuji Oil Co. Ltd., 1 Sumiyoshi-cho,
Izumisano, Osaka 598-0061, Japan
＊＊Novelty Materials Research Institute, Hannan R＆D Center, Fuji Oil Co. Ltd.,
1 Sumiyoshi-cho, Izumisano, Osaka 598-0061, JapanReceived January 19, 1999; Accepted April 30, 1999
Four-week-old rats were fed on diets containing either no dietary fiber (DF) or a DF source (WSSF, ISF or cellulose) for 4 weeks. The DF level was adjusted to 5％. The WSSF diet contained 3％ and 2％, respectively, of WSSF and cellulose. No rat in any group experienced diarrhea, and none of the experimental diets suppressed the growth of rats, the apparent absorption of major nutrients being almost 100％. However, the rate of degradation of DF during the digestive process was significantly different (p<0.05, cellulose, 23.6％; WSSF with cellulose, 64.5％ (WSSF degradation only was 91.8％); and ISF, 77.6％). The plasma and liver lipid levels were within normal ranges, although the liver cholesterol level in those rats fed on WSSF and ISF was significantly lower (p<0.05) than in those fed on cellulose. The cecal organic acid contents were in the order of WSSF>ISF>cellulose>DF-free. Furthermore, WSSF was effective in shortening the gastrointestinal transit time. The results indicate that WSSF seems to have favorable effects on the intestinal functions.
soybean; water-soluble fiber; dietary fiber; fermentation; rat
-5-
XynX, a Possible Exo-xylanase of Aeromonas caviae ME-1 that Produces
Exclusively Xylobiose and Xylotetraose from Xylan
Kengo USUI,1 Keiji IBATA,1 Tohru SUZUKI,1,・ and Keiichi KAWAI1,2
1Department of Biotechnology, Faculty of Agriculture, 2Molecular Genetics Research Center,
Gifu University, 1-1 Yanagido, Gifu 501-1193, JapanReceived January 20, 1999; Accepted April 16, 1999
A gene, xynX, encoding a novel xylanase, was cloned from Aeromonas caviae ME-1. This gene encoded an enzyme that was constituted of 334 amino acid residues (38,580 Da) and was similar in sequence to Family 10 (Family F) β-1,4 endo-xylanases. XynX produced only xylobiose and xylotetraose from birch wood xylan, and xylotriose, xylopentaose, and higher oligosaccharides were not detected in the TLC analysis. We designated it as X2/X4-forming xylanase. This enzyme does not have transglycosylation activity. These data suggested that this enzyme is a possible exo-xylanase. According to homology modeling, the enzyme has a ring-shaped (α/β)8 barrel (TIM barrel) structure, typical of Family 10 endo-xylanases, with the extraordinary feature of a longer bottom-loop structure.
exo-xylanase; Aeromonas caviae ME-1; Family 10; X2/X4-forming xylanase; gene cloning
-6-
New Method for Determining the Sugar Composition of Glycoproteins,
Glycolipids, and Oligosaccharides by High-performance
Liquid Chromatography
Shoichi YASUNO,・ Kazuko KOKUBO,＊ and Masugu KAMEI
Sugiyama Chemical and Industrial Laboratory, 11 Kagetori-cho, Totsuka-ku, Yokohama,
Kanagawa 245-0064, Japan
＊Honen Corporation, 1-2-3 Ohtemachi, Chiyoda-ku, Tokyo 100-0004, JapanReceived January 26, 1999; Accepted April 28, 1999
A new method is reported that can be performed within a single vessel to analyze the composition of aldose, hexosamine, and sialic acid residues of glycoproteins, glycolipids, and oligosaccharides. Glycoconjugates are treated with sialidase or subjected to mild acid hydrolysis, before being treated with N-acetylneuraminic acid aldolase to convert the free sialic acid residues to their corresponding N-acylmannosamines. The reaction mixture is then successively subjected to acid hydrolysis (in order to produce monosaccharides), N-acetylation, and conversion with ρ-aminobenzoic acid ethyl ester (ABEE). The ABEE-converted monosaccharides are simultaneously determined by reverse-phase high-performance liquid chromatography. Determination of the sugar compositions of bovine fetuin, II3NeuGcα-LacCer, and 3′-sialyllactose with this method was found to be highly accurate. Linearity of the peak area vs. the amount of bovine fetuin ranged from 1 to 50 μg in all ABEE-converted monosaccharides. With a slight modification to this method, sialic acid residues can be separately determined as NeuAc and NeuGc. This novel method and its modified version are used to demonstrate the sugar compositions of α1-acid glycoproteins from several sources.
ρ-aminobenzoic acid ethyl ester; sialic acid; α1-acid glycoprotein; monosaccharide analysis
-7-
A Simple Purification Method and Morphology and Component Analyses
for Carotovoricin Er, a Phage-tail-like Bacteriocin from the Plant
Pathogen Erwinia carotovora Er
Anh Hoa NGUYEN,1 Toshio TOMITA,1 Morihiko HIROTA,1 Tsuruji SATO,2
and Yoshiyuki KAMIO1,・1Laboratory of Applied Microbiology, and 2Division of Electron Microscopy, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
Received February 2, 1999; Accepted April 22, 1999
Carotovoricin Er has been isolated as a phage-tail-like bacteriocin from the plant pathogen Erwinia carotovora Er [Kamimiya, S. et al., (1977), Agric. Biol. Chem. 41, 911--912]. However, the fine morphology and structural composition of carotovoricin Er remained to be studied because a large amount of contracted carotovoricin Er were present in the bacteriocin preparations so far obtained. To obtain intact carotovoricin Er and its major parts, we developed simple and efficient purification methods including the use of sucrose density gradient centrifugation in the presence of 10--20％ (v/v) ethanol. Electron microscopy for the purified carotovoricin Er showed the presence of a novel antenna-like structure at the proximal end of the phage-tail-like particle, which consisted of a sheath-and-core part, a baseplate, and tail fibers. Contracted sheath and inner core were purified as hollow cylindrical structures with longitudinal lengths of 69 and 174 nm, respectively, and tail fibers were purified as a fibrous structure with length of 63 nm. SDS-polyacrylamide gel electrophoresis showed the presence of single major proteins of 50, 20, and 68 kDa in the isolated sheath, core, and tail fiber, respectively. Three other minor proteins of 46, 44, and 35 kDa were also identified as the structural proteins of carotovoricin Er, which may be the candidate proteins for the antenna-like and the base plate structures. Thus carotovoricin Er consists of at least 6 protein components.
Erwinia carotovora Er; carotovoricin Er; phage-tail-like bacteriocin; sucrose density gradient centrifugation; electron microscopy
-8-
Inhibition of DNA Topoisomerase I by Dihydrotanshinone I,
Components of a Medicinal Herb Salvia miltiorrhiza Bunge
Dong-Sun LEE,1,・ Sang-Han LEE,1 Gi-Seok KWON,2 Hong-Kum LEE,3
Joo-Hyung WOO,1 Jong-Guk KIM,1 and Soon-Duck HONG11Department of Microbiology, College of Natural Science, Kyungpook National University,
Taegu, 702-701, Korea
2Department of Life Resource Science, College of Natural Science, Andong National University,
Andong, 760-749, Korea
3Marine Microbiology Research Laboratory, Biological Oceanography Division, Korea Ocean Research ＆
Development Institute (KORDI), Ansan 1270, 425-600 KoreaReceived February 12, 1999; Accepted April 23, 1999
Dihydrotanshinone I induced topoisomerase I-mediated DNA cleavage in vitro as strongly as camptothecin, but topoisomerase II-mediated DNA cleavage was not affected. In a DNA relaxation assay using calf thymus DNA topoisomerase I and supercoiled pBR322 plasmid DNA, dihydrotanshinone I reduced topoisomerase I-mediated DNA relaxation in a dose-dependent manner. Heat treatment (65°C) of the reaction mixture containing dihydrotanshinone I and topoisomerase I resulted in a substantial reduction in DNA cleavage, suggesting topoisomerase I and dihydrotanshinone I may form a reversible cleavable complex to induce DNA damage. A DNA unwinding assay using T4 DNA ligase showed that dihydrotanshinone I is a very weak DNA intercalator. These results suggest that dihydrotanshinone I inhibits the catalytic activity of topoisomerase I by the formation of a cleavable complex and at least in part through the intercalation into DNA.
dihydrotanshinone I; DNA topoisomerase I inhibitor; DNA intercalator
-9-
Chemo-enzymatic Synthesis of both Enantiomers of myo-Inositol
1,3,4,5-tetrakisphosphate
Kurt LAUMEN and Oreste GHISALBA
Novartis Pharma AG, Core Technology Area, CH-4002 Basel, Switzerland
D-Ins(1,3,4,5)P4 and unnatural L-Ins(1,3,4,5)P4 were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.
D-myo-inositol 1,3,4,5-tetrakisphosphate; L-myo-inositol 1,3,4,5-tetrakisphosphate; Candida antarctica; lipase; enzymatic esterification
-10-
Production of Vitamin B6 in Rhizobium
Masaaki TAZOE,・ Keiko ICHIKAWA and Tatsuo HOSHINO
Department of Applied Microbiology, Nippon Roche Research Center, Kamakura,
Kanagawa 247-8530, JapanReceived February 17, 1999; Accepted April 30, 1999
The production of vitamin B6 was studied in about 1,590 bacterial isolates from soil, and an isolate, 28-21, identified as Rhizobium leguminosarum was obtained as a vitamin B6 high producer. Then, the production of vitamin B6 by commercially available Rhizobium strains was examined, and many of the tested strains excreted large amounts of vitamin B6 into the culture broth. The best producer of vitamin B6 was R. meliloti IFO 14782, which produced 51 mg per liter. Media study for the vitamin B6 production was done with R. meliloti IFO 14782; the strain was able to excrete 84 mg of vitamin B6 per liter, 79 mg per liter of which was pyridoxol.
vitamin B6 production; Rhizobium
-11-
Effects of Rat Fetuin on Stimulation of Bone Resorption in the Presence
of Parathyroid Hormone
Osamu NAKAMURA,＊ Jamil Ahsan KAZI,＊＊ Tomokazu OHNISHI, Naokatu ARAKAKI,＊＊＊
Qing SHAO,＊＊＊＊ Takehiro KAJIHARA, and Yasushi DAIKUHARA・Department of Biochemistry, Kagoshima University Dental School, 35-1 Sakuragaoka-8,
Kagoshima 890-8544, JapanReceived February 22, 1999; Accepted April 13, 1999
Rat fetuin, which is the rat counterpart of human α2-HS glycoprotein and bovine fetuin, is only detectable in calcified tissues such as bone matrices and dentin, and bone cells such as osteoblasts and osteocytes immunohistochemically. The effect of this protein on bone resorption was examined to study its physiological role in bone metabolism. Rat fetuin increased bone resorption in the presence of low concentrations of parathyroid hormone (PTH), but it had no activity on bone resorption without PTH. The increase in bone resorption by PTH and PTH plus rat fetuin was inhibited by the addition of chymostatin, an inhibitor for cathepsin L. Moreover, we found that when type I collagen from rat was preincubated with rat fetuin, the digestion of rat type I collagen by cathepsin L was increased. These findings suggest that rat fetuin present in bone matrix is important in bone resorption.
α2-HS glycoprotein; bone resorption; cathepsin L; parathyroid hormone; rat fetuin
-12-
Structural Properties of Recombinant Ovalbumin and Its Transformation
into a Thermostabilized Form by Alkaline Treatment
Yasuhiro ARII, Nobuyuki TAKAHASHI, Eizo TATSUMI,＊ and Masaaki HIROSE・
The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
Received February 24, 1999; Accepted April 16, 1999
The recombinant ovalbumin produced in Escherichia coli was purified from the cytoplasmic fraction and analyzed for its chemical and conformational properties. The recombinant ovalbumin displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as egg white ovalbumin. As in the egg white protein, four cysteine sulfhydryls and one cystine disulfide were contained in the recombinant protein, according to the results of amino acid analyses; the disulfide bond was found by a peptide mapping analysis to correspond to the native cystine, Cys73-Cys120. According to a gel electrophoresis analysis, the presence of the disulfide bond was accounted for by specific oxidation of the corresponding cysteine residues during purification of the cytoplasmic protein. Unlike the identity in the conformational and peptide structures, none of the post-translational modifications (N-terminal acetylation, phosphorylation, and glycosylation) that are known with egg white ovalbumin were detected in the recombinant protein. The recombinant ovalbumin was transformed into a thermostabilized form in a similar manner to the transformation of egg white protein into S-ovalbumin; alkaline treatment increased the temperature for thermostability by 8.7°C. These data strongly suggest that the post-translational modifications of ovalbumin are not related to the formation mechanism for S-ovalbumin.
ovalbumin; recombinant ovalbumin; S-ovalbumin
-13-
Deterioration of Tolerance to Hydrophobic Organic Solvents
in a Toluene-Tolerant Strain of Pseudomonas putida
under the Conditions Lowering Aerobic Respiration
Kasumi NOGUCHI, Norihiko TSUKAGOSHI, and Rikizo AONO・
Department of Biological Information, Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, JapanReceived February 26, 1999; Accepted April 16, 1999
The growth curve (increase in the number of viable cells) of a toluene-tolerant strain Pseudomonas putida Px51T was not reproducible in the presence of harmful organic solvents, such as p-xylene and toluene. The survival often fluctuated the during late exponential phase of growth. The repetitive growth was obtained by maintaining pO2 20--40％ (v/v) in the culture flask. However, even under these aerobic conditions, the cells starved for a carbon source were killed by exposure to harmful solvents. The tolerance to organic solvents was lowered greatly by treatment with a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP), or an electron transport chain inhibitor, sodium azide. Px51T treated with CCCP lost tolerance to a wide variety of organic solvents with log Pow of 2.6--4.2, which the organism usually tolerates. These results indicate that the solvent tolerance of Px51T depends upon on energy produced by aerobic respiration.
Pseudomonas putida; organic solvent; organic solvent tolerance; aerobic respiration
-14-
cDNA Subtractive Cloning of Genes Expressed during Early Stage
of Appressorium Formation by Magnaporthe grisea
Takashi KAMAKURA,・・ Jin-zhong XIAO,・ Woo-Bong CHOI,・ Takamasa KOCHI,＊
Syuichi YAMAGUCHI,＊ Tohru TERAOKA,＊ and Isamu YAMAGUCHIMicrobial Toxicology Laboratory, The Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198, Japan
＊Tokyo University of Agriculture and Technology, Fucyu, Tokyo 183-8509, JapanReceived March 1, 1999; Accepted April 16, 1999
The conidial germ tube of the fungus Magnaporthe grisea differentiates an infection-specific structure, an appressorium, for penetration into the host plant. Formation of the appressorium is also observed on synthetic solid substrata such as polycarbonate. We found that a plant lectin, concanavalin A, specifically suppressed the appressorium formation without affecting the germling adhesion if it was applied within 2--3 hours after germination. Standing on the result, we constructed a cDNA library that represents the early stage of germ tube development and/or appressorium formation from the 2.5-hour-old germ tubes using a cDNA subtraction strategy by the combination of the biotin labeled driver method and adapter-primed PCR method. Out of 686 colonies of the library, 158 distinct clones′ nucleotide sequences were partially analyzed. Some clones′ expression patterns were detected by RT-PCR and from those results, our library seemed to well represent the objective developmental stage of M. grisea.
Magnaporthe grisea; Pyricularia oryzae; appressorium formation; cDNA subtraction; differential library
-15-
Isolation and Characterization of the Gene Conferring Thiamine-inducible
Expression from Saccharomyces cerevisiae
Yoichiro SHIBA,・ Chiho ONO, Kimihisa ICHIKAWA, Nobufusa SERIZAWA,
and Hiroji YOSHIKAWABiomedical Research Laboratories, Sankyo Co. Ltd., 389-4 Aza-Ohtsurugi, Shimokawa,
Izumimachi, Iwaki, Fukushima 971-8183, JapanReceived March 4, 1999; Accepted May 6, 1999
The production level of CPY in Saccharomyces cerevisiae KS58-2D/pCY303 was drastically decreased when thiamine was not added to the culture medium. We isolated and characterized the mutants that could produce CPY even though thiamine was absent from the medium. Using complementation screening in the mutants obtained, we isolated a gene that was involved in the thiamine-inducible expression, TIE1, which corresponded to the YDR325w ORF on chromosome IV. The predicted protein sequence of TIE1 did not have significant homology to proteins from public databases. The disruption of the TIE1 gene caused two phenotypes, increase of expression level in thiamine-free medium and ethanol sensitivity. This increase in thiamine-free medium was also observed in the expression under the control of ENO1 or ADH1 promoter in addition to the GAL10 promoter, suggesting that the TIE1 protein is associated with a similar kind of transcriptional mechanism regulated by thiamine.
Saccharomyces cerevisiae; thiamine-inducible expression; ethanol sensitivity; TIE1
-16-
Six Diacylated Anthocyanins from the Storage Roots
of Purple Sweet Potato, Ipomoea batatas
Norihiko TERAHARA,・ Takashige SHIMIZU,＊ Yoshiaki KATO,＊ Mikio NAKAMURA,＊
Tamio MAITANI,＊＊ Masa-atsu YAMAGUCHI, and Yukihiro GODA＊＊Department of Food Science and Technology, College of Horticulture, Minami-Kyushu University,
Takanabe, Miyazaki 884-0003, Japan
＊San-Ei Gen F.F.I. Inc., Sanwa-cho, Toyonaka, Osaka 561-8588, Japan
＊＊National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, JapanReceived March 11, 1999; Accepted May 7, 1999
Eight acylated anthocyanins were isolated from the storage roots of the purple sweet potato, Ipomoea batatas cv Yamagawamurasaki, which is the source of the food colorant ``purple sweet potato color.′′ Of these, six pigments were identified as diacylated anthocyanins, cyanidin and peonidin 3-O-(6-O-(E)-caffeyl-2-O-(6-O-acyl-||β - D - glucopyranosyl) - β - D - glucopyranoside) - 5 - O - β - D - gluco||pyranosides, in which each acyl subsutituent was a p-hydroxybenzoyl, (E)-caffeyl or (E)-ferulyl residue, mainly by NMR analyses.
Ipomoea batatas; Convolvulaceae; diacylated anthocyanin; purple sweet potato color; NMR
-17-
Interaction of Paratropomyosin with β-Connectin and Its 400-kiloDalton
Fragment from Chicken Skeletal Muscle as Influenced
by the Calcium Ion Concentration
Sha FEI, Minoru YAMANOUE,＊,・ and Takahide OKAYAMA＊
Graduate School of Science and Technology, and ＊Department of Biofunctional Chemistry,
Faculty of Agriculture, Kobe University, Kobe, Hyogo 657-8501, JapanReceived March 15, 1999; Accepted May 6, 1999
The binding of paratropomyosin to β-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of β-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10－7 M to 10－5 M and markedly suppressed above 10－4 M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of β-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.
paratropomyosin; β-connectin; 400-kDa fragment; meat tenderization; postmortem ageing
-18-
Green-Tissue-Specific Expression of a Reconstructed cry1C Gene Encoding
the Active Fragment of Bacillus thuringiensis δ-Endotoxin
in Haploid Tobacco Plants Conferring Resistance to Spodoptera litura
Nikolai Kirilov CHRISTOV,＊,・ Hiromasa IMAISHI,＊＊ and Hideo OHKAWA＊,・・
＊The Graduate School of Science and Technology, Bioscience Division, and
＊＊Center for Cooperative Research and Development Kobe University,
1-1 Rokkodai, Nada, Kobe 657-8501, JapanReceived March 18, 1999; Accepted May 9, 1999
The DNA sequence of a truncated cry1C gene encoding the active fragment of Bacillus thuringiensis (Bt) δ-endotoxin was fully reconstructed by introduction of silent mutations. Each of the truncated wild type and the synthetic genes encoding the active fragment of the protoxin was introduced into haploid tobacco plants under the control of the rbcS promoter. To facilitate selection of transgenic tobacco plants with high insecticidal activity, a fusion gene encoding both rat CYP1A1 cytochrome P450 and yeast NADPH-P450 oxidoreductase was cotransformed with the wild type cry1C gene. The synthetic gene elevated the levels of Cry1C protein and the mRNA in transgenic tobacco plants as well as mortality in Spodoptera litura larvae. The Cry1C protein was accumulated mainly in the leaf tissues of the transgenic tobacco plants. The results reported here imply that the green-tissue-specific expression of the synthetic cry1C gene is useful for the control of S. litura which was rather resistant to the other types of Bt toxins.
Bacillus thuringiensis; cry1C; insect resistance; Spodoptera; transgenic plants
-19-
Heterologous Expression and Product Identification
of Colletotrichum lagenarium Polyketide Synthase Encoded
by the PKS1 Gene Involved in Melanin Biosynthesis
Isao FUJII, Yuichiro MORI, Akira WATANABE, Yasuyuki KUBO,1
Gento TSUJI,1 and Yutaka EBIZUKA・Graduate School of Pharmaceutical Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-0033, Japan
1Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, JapanReceived March 18, 1999; Accepted May 11, 1999
The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible α-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN.
Colletotrichum lagenarium; melanin biosynthesis; polyketide synthase; heterologous expression; 1,3,6,8-tetrahydroxynaphthalene
-20-
Stereoselective Syntheses of (－)-Podorhizol Lignan and its Derivatives:
erythro and threo Preferential Aldol Condensation of Potassium Enolate
from γ-Butyrolactone with Alkoxybenzaldehyde
Satoshi YAMAUCHI,・ Mitsuo MACHI, and Yoshiro KINOSHITA
College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan
Received April 7, 1999; Accepted May 7, 1999
(－)-Podorhizol (1) was stereoselectively synthesized by||erythro preferential aldol condensation of 3,4,5-trimethoxy||||benzaldehyde with potassium enolate from (＋)-(R)-3-||(3,4-methylenedioxybenzyl)-4-butanolide (2) (erythro:threo＝85:15). Erythro selectivity was observed in the aldol condensation of many alkoxybenzaldehydes with potassium enolate from (＋)-γ-butyrolactone 2. However, benzaldehydes having methoxy groups at both the 2 and 6 positions gave threo selectivity in the aldol condensation with potassium enolate from (＋)-γ-butyrolactone 2.
lignan; γ-butyrolactone; dibenzyl-γ-butyrolactone; podorhizol; aldol condensation
-21-
Note
Toxicity of Cadmium Particle Dust in Bacterial Cells
Naoto YOSHIDA,・1 Terutoyo YOSHIDA,2 and Kihachiro OGAWA1
1Department of Biological Resource Sciences, and
2Department of Fisheries, Faculty of Agriculture, Miyazaki University,
1-1 Gakuen Kibanadai-Nishi, Miyazaki-shi 889-2192, JapanReceived January 20, 1999; Accepted April 12, 1999
When Thiobacillus intermedius 13--1, Escherichia coli JM109, and Agrobacterium radiobacter IFO12665b1 were cultured in LB liquid medium containing 0.03 g or 0.1 g of cadmium particle dust, growth was strongly inhibited. Expose of cells to cadmium particle resulted in an increase in pH and concentration of ammonium ions. No cadmium particle attachment to the outer wall of T. intermedius or cell wall disruption was observed by transmission electron microscopy.
cadmium dust; Thiobacillus; bacteria; particle; toxicity
-22-
Note
Effects of Glucose on Lipase Activity
Wakako TSUZUKI,1・ Yoshiaki KITAMURA,1 Tateo SUZUKI,1
and Tamio MASE21National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries,
Kannondai 2-1-2 Tsukuba, Ibaraki 305-8642, Japan
2Amano Pharmaceutical Co. Ltd., Nishiki 1-2-7, Naka-ku, Nagoya, Aichi 481-8543, JapanReceived January 22, 1999; Accepted April 13, 1999
To establish the utility of lipase as a biocatalyst, the effects of glucose on the hydrolysis activities of lipase were investigated. Among 13 kinds of lipase from microorganisms, 6 lipases were inhibited in hydrolysis up to 50％ of the original activities by 10 mM glucose. The activities of other microbial lipases and 2 kind of porcine pancreatic lipases were not affected by the addition of glucose. Six lipases that were sensitive to glucose were modified by a synthetic detergent. After they were converted to modified lipases, they were not inhibited by glucose. Even at 20 mM glucose, each modified lipase retained more than 95％ activity compared with that in the absence of glucose. In the modified lipase, the detergent attached to the lipase molecule would disturb the access of glucose to the enzyme. To detect the interaction between lipase and glucose, the fluorescence of tryptophan was traced. The fluorescence intensities of lipases that were inhibited by glucose depended on the concentration of glucose, suggesting that glucose induced some structural change in the lipase molecule.
lipase; hydrolysis; inhibition; glucose
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Note
Fluorogenic Substrates for Cathepsin D
Hiroo YONEZAWA,＊ Tetsuya UCHIKOBA, Kazunari ARIMA,＊＊
and Makoto KANEDADepartment of Chemistry, Faculty of Science, Kagoshima University,
1-Korimoto, Kagoshima 890-0065, Japan
＊＊Department of Plant Biology, Plant Gene Expression Center,
800 Buchanan St. Albany, CA 94710 U.S.A.Received January 26, 1999; Accepted April 16, 1999
Fluorogenic substrates for cathepsin D; A-Tyr-Phe(NO2)-Leu-Leu (A; Ala-Arg-Pro-Lys-Pro-Leu-Leu-, Arg-Pro-Lys-Pro-Leu-Leu-, Pro-Lys-Pro-Leu-Leu-, Lys-Pro-Leu-Leu-, Pro-Leu-Leu-) and B-Phe(NO2)-Tyr-Leu-Leu (B; Arg-Pro-Lys-Pro-Leu-Leu-, Pro-Lys-Pro-Leu-Leu-, Lys-Pro-Leu-Leu-, Pro-Leu-Leu-) (Phe(NO2), p-nitrophenylalanine) were synthesized and digested by cathepsin D and pepsin. The fluorescence at 303 nm (excitation at 260 nm) was increased with the hydrolysis of the substrates. The minimum detectable cathepsin D concentrations for these substrates were 0.5--4 nM and pepsin concentrations were 0.1--0.8 nM except Pro-Leu-Leu-Tyr-Phe(NO2)-Leu-Leu under the following conditions: substrate concentration, 20 μM; measuring time, 3 min. The hydrolysis rate constants (kcat/Km) of B-Phe(NO2)-Tyr-Leu-Leu for cathepsin D were same or 2--3 times greater than A-Tyr-Phe(NO2)-Leu-Leu. On the other hand, those of B-Phe(NO2)-Tyr-Leu-Leu for pepsin were the same or 4--20 times greater than A-Tyr-Phe(NO2)-Leu-Leu. The hydrolysis rates of the substrates by both enzymes tend to increase with the increase of the peptide chain length. The best substrate for cathepsin D was Arg-Pro-Lys-Pro-Leu-Leu-Phe(NO2)-Tyr-Leu-Leu and its kcat/Km was 1.3 μM－1 s－1.
cathepsin D; measurement of protease activity; fluorogenic substrate
-24-
Note
Arg-Gingipain Inhibition and Anti-bacterial Activity Selective
for Porphyromonas gingivalis by Malabaricone C
Chikara SHINOHARA, Satoko MORI, Tomomi ANDO, and Tomoko TSUJI
Sagami Chemical Research Center, 4-4-1 Nishiohnuma, Sagamihara-shi, Kanagawa 229-0012, Japan
Received February 10, 1999; Accepted April 21, 1999
Effects of malabaricon C, isolated from nutmeg (Myristica fragrans), on Arg-gingipain activity and growth of several kinds of anaerobic and aerobic microorganisms were investigated. Malabaricone C irreversibly inhibited Arg-gingipain by 50％ at a concentration of 0.7 μg/ml and selectively suppressed Porphyromomas gingivalis growth.
malabaricone C; Arg-gingipain; Porphyromonas gingivalis; periodontal disease; Myristica fragrans
-25-
Note
Identification of the New Hydrocarbon (Z,Z)-1,6,9-Heptadecatriene
as the Secretory Component of Caloglyphus polyphyllae
(Astigmata: Acaridae)・
Nobuhiro SHIMIZU, Naoki MORI, and Yasumasa KUWAHARA＊
Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanReceived February 12, 1999; Accepted May 6, 1999
A new heptadecatriene was isolated from the acarid mite, Caloglyphus polyphyllae, as the major characteristic component which could be used to identify the species chemo-taxonomically. Its structure was elucidated as 1,6,9-heptadecatriene by partial hydrogenation and a subsequent GC/MS analysis of the dimethyldisulfide derivative, together with evidence of the terminal vinyl group and Z-configuration of double bonds that was provided by GC-FT/IR and NMR. The triene was identified as (Z,Z)-1,6,9-heptadecatriene by its synthesis and is revealed to be a new compound as a natural product.
Caloglyphus polyphyllae; hydrocarbon; (Z,Z)-1,6,9-heptadecatriene
-26-
Note
Viscometry of Curdlan, a Linear (1→3)-β-D-Glucan,
in DMSO or Alkaline Solutions
Hiroaki FUTATSUYAMA, Toshifumi YUI,＊ and Kozo OGAWA・
Research Institute for Advanced Science and Technology (RIAST), Osaka Prefecture University,
1-2 Gakuen-cho, Sakai, Osaka 599-8570, Japan
＊Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, JapanReceived February 17, 1999; Accepted April 21, 1999
A simple method to obtain the molecular weight of curdlan was found by viscometry in its alkaline or DMSO solution. DMSO was found to be the most appropriate solvent for measuring Mv of curdlan. Based on two Sakurada-Houvink equations, [η]＝KMa, which have been obtained so far in alkaline solutions, two sets of parameters of the equation in the DMSO solution, that is, K＝3.5×10－4, a＝0.65 and K＝1.6×10－4, a＝0.74 were introduced, respectively. Both parameter sets seemed to serve practically to measure the molecular weight of curdlan. In addition, using the equation obtained with several NaOH concentrations, the previous speculation that curdlan conformation changes from a rigid rod to random coil with alkaline concentration was confirmed.
curdlan; DMSO; viscosity; molecular weight
-27-
Note
Syntheses and Potato Tuber-inducing Activities
of Unnatural Long-chain OPC-9:0 and OPC-10:0
Hiroaki TOSHIMA, Yuko TAKANO, Akitami ICHIHARA, Yasunori KODA,＊
and Yoshio KIKUTA＊Department of Bioscience and Chemistry; ＊Department of Botany, Faculty of Agriculture,
Hokkaido University, Sapporo 060-8589, JapanReceived February 19, 1999; Accepted April 15, 1999
The unnatural long-chain OPCs, OPC-9:0 and OPC-10:0, were respectively synthesized from the ethyl esters of OPC-7:0 and OPC-8:0. C2-carbon elongation was achieved via alkylation of the enolate of tert-butyl acetate. The potato tuber-inducing activity of OPC-10:0, as well as OPC-8:0, -6:0 and -4:0, was similar to that of jasmonic acid. OPC-9:0 also exhibited weak tuber-inducing activity.
OPC; 12-oxo-PDA; jasmonic acid; tuber-inducing activity
-28-
Note
Effects of Recombinant Nitrophorin-2 Nitric Oxide Complex
on Vascular Smooth Muscle
Yuji KANEKO, Hideki SHOJO,＊ Masao YUDA, and Yasuo CHINZEI・
Department of Medical Zoology, School of Medicine, Mie University,
2-174 Edobashi, Tsu, Mie 514-0001, Japan
＊Department of Forensic Science, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, JapanReceived February 22, 1999; Accepted April 14, 1999
Nitrophorin-2, isolated from the salivary gland of the blood-sucking insect Rhodnius prolixus, is a nitric oxide (NO) binding protein. We investigated the effects of recombinant nitrophorin-2 NO complex on vascular smooth muscle. The course of relaxation was relative to released NO from recombinant nitrophorin-2 NO complex. Our data suggested nitrophorin-2 was tightly adhesive to the membranes to transport NO into the cell during the insect sting.
cGMP; hemoprotein; nitric oxide; Rhodnius prolixus; vasodilator
-29-
Note
Immunoaffinity Purification and Identification
of the Molecular Chaperone Calnexin
Tetsuro YAMASHITA,・ Emiko KIYOKI, Yasuhiro TOMITA, and Hideharu TAIRA
Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
Received February 22, 1999; Accepted April 16, 1999
We have developed a method for the immunoaffinity purification of calnexin, an endoplasmic reticulum molecular chaperone, and analyzed the molecular weight of purified calnexin using matrix-assisted laser adsorption ionization time of flight mass spectrometry (MALDI TOF-MS). Calnexin was thereby found to have a molecular weight of 66.1×103, which is nearly identical to the molecular weight estimated from the protein sequence.
calnexin; molecular chaperone; affinity purification; mass spectrometry
-30-
Note
Antimicrobial Activity of Monascus pilosus IFO 4520
against Contaminant of Koji
Isato KONO・ and Kunio HIMENO
Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama-shi,
Okayama 701-1296, JapanReceived February 22, 1999; Accepted May 10, 1999
Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5％ lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.
Monascus pilosus; beni-koji; antimicrobial activity; contaminant
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Note
Preparation of 4-(4′-Hydroxyanilino)-5-anilinophthalimide
and 4,5-Bis-(4′-hydroxyanilino)-phthalimide by Microbial Hydroxylation
Stefan WEIDNER,＊ Klaus GOEKE, Uwe TRINKS, Peter TRAXLER,＊＊
Katharina UCCI-STOLL, and Oreste GHISALBA・Novartis Pharma AG, Research, Core Technology Area, Expertise Bioreactions, 4002 Basel, Switzerland
＊Institute of Enzyme Technology, Heinrich-Heine-University D¨・ usseldorf, Research Center J¨・ ulich, 52426 J¨・ ulich, Germany
＊＊Oncology Research, Novartis Pharma Inc., 4002 Basel, SwitzerlandReceived March 8, 1999; Accepted May 6, 1999
A microbial screening indicated that two fungal strains, Beauveria bassiana DSM 1344＝ATCC 7159 and Cunninghamella elegans DSM 1908＝ATCC 9245, as well as four bacterial strains belonging to the genus Streptomyces were able to hydroxylate 4,5-dianilinophthalimide (DAPH, CGP52411) to 4-(4′-hydroxyanilino)-5-anilinophthalimide. Cunninghamella elegans DSM 1908 turned out to be the most active biocatalyst and was also able to form the dihydroxy derivative, 4,5-bis(4′-hydroxyanilino)phthalimide. The reaction for the monohydroxylated biotransformation product was carried out on a preparative scale, and||the culture conditions for the formation of 4-(4′-hydroxy||||anilino)-5-anilinophthalimide with this strain were op||timized.
PTK-inhibitors; 4-(4′-hydroxyanilino)-5-anilinophthalimide; 4,5-bis-(4′-hydroxyanilino)-phthalimide; microbial hydroxylation
-32-
Note
Differentiation in a Rat PC12 Cell Line Induced by Ostruthin
and (－)-Bornyl Ferulate, Constituents of a Chinese Herbal Medicine
Jianhua QI, Makoto OJIKA, and Youji SAKAGAMI・
Graduate School of Bioagricultural Sciences, Nagoya University,
Chikusa-ku, Nagoya 464-8601, JapanReceived March 10, 1999; Accepted May 8, 1999
A search for neuritogenic compounds in Chinese herbs resulted in the isolation of two known substances, ostruthin and (－)-bornyl ferulate, from Notopterygium incisum (and/or N. forbesii). Both compounds induced comparable neurite-like structures in 20％ of rat PC12 cells at 2 μg/ml, and showed cytotoxicity at concentrations higher than 3 μg/ml.
ostruthin; (－)-bornyl ferulate; neuritogenic; Chinese herbal medicine; PC12 cells
-33-
Note
Determination of a Small Quantity of Cystine in the Presence
of a Large Amount of Cysteine
Shuzo YAMAGATA・ and Tomonori IWAMA
Department of Biotechnology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
Received March 23, 1999; Accepted May 6, 1999
A procedure is described to precisely determine a very small amount of cystine in the presence of a large amount of cysteine. After completely modifying cysteine with N-ethylmaleimide, the remaining reagent was reacted with DL-homocysteine. Cystine was determined, after being reduced with dithiothreitol, by the reaction with ninhydrin carried out under acidic conditions. The procedure makes it possible to precisely determine the amount of cystine present with cysteine in a concentration ratio of 1:2,000. By employing this procedure, auto-oxidation of cysteine to cystine in a mixture for the L-cysteine α,β-elimination reaction was investigated.
cystine determination; cysteine; cystathionine; ninhydrin
-34-
Note
Simultaneous Determination of Lipid Hydroperoxides
by HPLC-Post Column Systems
Kazuaki AKASAKA,・ Hirotaka OHTA, Yoshimi HANADA, and Hiroshi OHRUI・
Department of Applied Life Science, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, JapanReceived March 24, 1999; Accepted May 7, 1999
Two HPLC-post column systems, a conventional-size system and a semi-micro system, for the simultaneous determination of lipid hydroperoxides were developed. The hydroperoxides of free fatty acids, phosphatidylcholines, triacylglycerols and cholesterol esters were individually determined at the pmol level with good reproducibility by using gradient elution and post-column detection with diphenyl-1-pyrenylphosphine.
lipid hydroperoxide; diphenyl-1-pyrenylphosphine; semi-micro high-performance liquid chromatography; post-column high-performance liquid chromatography
-35-
Note
Anthocyanins in The Dark Purple Anthers of Tulipa gesneriana: Identification
of Two Novel Delphinidin 3-O-(6-O-(Acetyl-α-Rhamnopyranosyl)-
β-Glucopyranosides)
Masayoshi NAKAYAMA,1 Masa-atsu YAMAGUCHI,2 Osamu URASHIMA,3 Yukiko KAN,4
Yuko FUKUI,5 Yuichi YAMAGUCHI,6 and Masaji KOSHIOKA1,・1Department of Floriculture, National Research Institute of Vegetables, Ornamental Plants and Tea (NIVOT),
Ministry of Agriculture, Forestry and Fisheries, 360 Kusawa, Ano, Mie 514-2392, Japan
2Minami-Kyushu University, Takanabe, Miyazaki 884-0003, Japan
3Toyama Agricultural Research Center, 288 Gorohmaru, Tonami, Toyama 939-1327, Japan
4Suntory Institute for Bioorganic Research (SUNBOR), 1-1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan
5Institute for Fundamental Reserch, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto, Osaka 618-8503, Japan
6Department of Tea Processing Technology, NIVOT, 2769 Kanaya, Kanaya, Shizuoka 428-8501, JapanReceived April 13, 1999; Accepted May 12, 1999
Two novel anthocyanins, delphinidin 3-O-(6-O-(2-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside) and delphinidin 3-O-(6-O-(3-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside), were identified from the anthers of Tulipa gesneriana. These and delphinidin 3-O-(6-O-(α-rhamnopyranosyl)-β-glucopyranoside) made up over 80％ of the anthocyanin content in the dark purple anthers and could be responsible for the intense color of the anthers.
Tulipa gesneriana; dark purple anther; delphinidin 3-O-(6-O-(α-rhamnopyranosyl)-β-glucopyranoside); delphinidin 3-O-(6-O-||(2 - O - acetyl - α - rhamnopyranosyl) - β - glucopy||ranoside); delphinidin 3-O-(6-O-(3-O-acetyl-α-rhamnopyranosyl)-β-glucopyranoside)
-36-
Preliminaly Communication
Identification of Blue Pigment Formed in a D-Xylose-Glycine Reaction System
Fumitaka HAYASE,＃＊ Yota TAKAHASHI,＊ Shigeru TOMINAGA,・ Masayo MIURA,・
Toshiharu GOMYO,・ and Hiromichi KATO§＊Department of Agricultural Chemistry, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
Bioscience Institute, Meiji Seika Kaisya, Ltd, 5-3-1 Chiyoda, Sakado-shi 350-0214, Japan
Kagawa Nutritional College, 3-9-21 Chiyoda, Sakado-shi 350-0214, Japan
§Department of Food Science, Otsuma Women′s University, Chiyoda-ku, Tokyo 102-8357, JapanReceived April 12, 1999; Accepted May 25, 1999
D-Xylose (1 M), glycine (0.1 M), and sodium hydrogencarbonate (0.1 M) were dissolved in aqueous 60％ ethanol at pH 8.1 and left at 26.5°C for 2 days in a dark room under nitrogen displacement. Blue pigment was isolated and purified from the blue solution by anionic exchange and gel filtration chromatographies. Blue pigment which was designated Blue-M1 (blue Maillard reaction intermediate-1) was||identified as 5-{[1,4-(dicarboxymethyl)-5-(2,3-dihydrox||||ypropyl) - 2 - pyrrolo[3,2 - b]pyrrolyl]methine} - 1,4 - (dicarbox||||ymethyl) - 2 - (1,2,3 - trihydroxypropyl) - pyrrolo[3,2 - b]pyrroly||lium. Blue-M1 is supposed to be a dimer of yellow colored pyrrolopyrrole-2-carboxaldehyde compounds. Blue-M1 that reacts readily to yellow compounds has a polymerizing activity, suggesting it is an important Maillard reaction intermediate through the formation of melanoidins.
Maillard reaction; xylose; glycine; blue compound; browning