Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 63 , Issue 7
Showing 1-32 articles out of 32 articles from the selected issue
Analytical Chemistry Regular Paper
Analytical Chemistry Note
Organic Chemistry Regular Papers
  • Tsutomu SATO, Tsutomu HOSHINO
    1999 Volume 63 Issue 7 Pages 1171-1180
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Site-directed mutagenesis experiments were carried out to identify the responsibility of the eight QW motifs for the reaction catalyzed by squalene-hopene cyclase (SHC). Alterations of the conserved tryptophans, which are responsible for the stacking structure with glutamine, into aliphatic amino acids gave a significantly lower temperature for the catalytic optimum as for the mutageneses of QW motifs 4, 5a and 5b, which are specifically present in SHCs. However, there was no change in the optimal temperatures of the mutated SHCs targeted at the other five motifs 1, 2, 3, 5c and 6. Thus, reinforcement against heat denaturation can be proposed as a function of the three QW motifs 4, 5a and 5b, but no function could be identified for the QW motifs 1, 2, 3, 5c and 6, although they are commonly found in all the families of prokaryotic SHCs and eukaryotic oxidosqualene cyclases. On the other hand, the three conserved tryptophans of W169, W312 and W489, which are located inside the putative central cavity and outside the QW motifs, were identified as components of the active sites, but also had a function against thermal denaturation. The other two tryptophan residues of W142 and W558, which are located outside the QW motifs, were found not to be active sites, but also had a role for stabilizing the protein structure. It is noteworthy that the mutants replaced by phenylalanine had higher temperatures for the catalytic optimum than those replaced by aliphatic amino acids. The catalytic optimal pH values for all the mutants remained unchanged with an identical value of 6.0.
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  • Hiroyuki TAZAKI, Takaomi HAYASHIDA, Mayumi ITO, Sayaka MINOSHIMA, Tats ...
    1999 Volume 63 Issue 7 Pages 1238-1241
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      The new lignan derivative, erimopyrone, was isolated from the liverwort, Moerckia erimona. Its structure was established as [1R, 2S]-1(6-carboxy-2-oxo-2H-4-pyranyl)- 6,7-dihydroxy-1,2-dihydro-2,3-naphthalenedicarboxylic acid by spectroscopic methods.
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  • Masakazu OKADA, Hisashi MIYAGAWA, Tamio UENO
    1999 Volume 63 Issue 7 Pages 1253-1260
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      AK-toxin I, a host-specific toxin to Japanese pear (Pyrus serotina), was synthesized as its methyl ester from three precursor fragments: conjugated diene-carboxylic acid, chiral epoxyalcohol and β-methylphenylalanine. The epoxyalcohol fragment was derived from D-fructose, in which effective homologation of the hemiacetal carbon to alkyne by using dimethyl 1-diazo-2-oxopropylphosphonate was the key reaction. The diene-carboxylic acid fragment was prepared by repeated Wittig reactions, and was combined with the epoxyalcohol fragment by the Stille reaction. Esterification of the combined product with the stereochemically-pure β-methylphenylalanine fragment afforded the target compound. This method was used to prepare the methyl ester of tritium-labeled AK-toxin I with a specific radioactivity of 213 GBq/mmol.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Jiro OAKI, Kayoko NAKAHARA, Masahiro TAMURA, Hideo OKAI
    1999 Volume 63 Issue 7 Pages 1156-1159
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      To search for techniques of simplified peptide synthesis, benzyloxycarbonyl chymotrypsin was prepared by a water-soluble acylating reagent and used to make Ac-Phe-Lys, an artificial peptide sweetener, which was selected as a target compound. As a result of using chemically modified chymotrypsin, Lys can be coupled directly with Ac-Phe and Ac-Phe-Lys made virtually in one step. Moreover, the total yield from preparation and purification steps for Ac-Phe-Lys was 13%. The value corresponds to that of the chemical synthesis method. On the contrary, enzymatic synthesis using native chymotrypsin cannot reach the level of the new method. It is expected that the method is more effective for simplified peptide synthesis as compared with other methods, especially on a large scale.
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  • Hiroyuki KUMETA, Tamotsu HOSHINO, Takako GODA, Tomohiko OKAYAMA, Takay ...
    1999 Volume 63 Issue 7 Pages 1165-1170
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      An extracellular metalloprotease named No.114 protease is one of the major secretions of a psychrotrophic bacterium, Pseudomonas fluorescens 114, the cold-adaptation mechanism of which has not been identified. In this study, we purified and cloned No.114 protease, which is a single polypeptide having a molecular mass of 47 kDa. This protease contains a zinc-binding motif (HEXXHXUGUXH: X, arbitrary amino acid; U, bulky hydrophobic amino acid), glycine-rich repeats (GGXGXD) and no cysteine residue, which are the features specifically found in serralysin subfamily. No.114 protease has its maximum activity at the temperature of 35-40°C, which is about 20°C lower than that of a serralysin from a mesophilic bacterium, Pseudomonas aeruginosa. All these results imply that No.114 protease from this psychrophilic bacterium is a unique member of the serralysin group characterized by a low optimal temperature.
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  • Pingyi ZHANG, Lina ZHANG, Shuyao CHENG
    1999 Volume 63 Issue 7 Pages 1197-1202
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      α-Glucan (L-FV-II) and β-glucan (L-FV-I) were isolated from fruiting bodies of Lentinus edodes by extraction with 5% NaOH/0.05% NaBH4, then precipitation with 1 M acetic acid. The analysis results of Smith degradation, high-performance liquid chromatography (HPLC), infrared spectra (IR), 13C NMR spectra, and optical rotation indicated that L-FV-II is α-(1→3)-D-glucan with slight (1→6) branch linkages, and L-FV-I is β-(1→3)-D-glucan containing 10% protein, and with branches. The weight-average molecular weight, Mw, number-average molecular weight, Mn, radii of gyration, <s2>1/2, and second virial coefficients, A2, of L-FV-II in aqueous 0.5 M urea/0.5 M NaOH (obtained from diluting its solution in 1.0 M urea/1.0 M NaOH) and 0.25 M LiCl in dimethylsulfoxide (DMSO/0.25 M LiCl) were measured by light scattering, membrane osmometry, and size exclusion chromatography (SEC). The values of Mw and Mn for L-FV-II in 0.5 M urea/0.5 M NaOH are 24.1×104 and 10.9×104, respectively, similar to those in DMSO/0.25 M LiCl. It was proved that strong intermolecular hydrogen bonds exist in L-FV-II, resulting in its water-insolubility, but there are not any aggregates or multiple-helix structure for L-FV-II in DMSO/0.25 M LiCl. The chain of α-(1→3)-D-glucan is more extended in DMSO/0.25 M LiCl than that in the aqueous solution.
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  • Tomohisa OGAWA, Chihiro ISHII, Daiji KAGAWA, Koji MURAMOTO, Hisao KAMI ...
    1999 Volume 63 Issue 7 Pages 1203-1208
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5′ and 3′ untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS=2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.
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  • Nobuo YAMASHITA, Toru MOTOYOSHI, Akira NISHIMURA
    1999 Volume 63 Issue 7 Pages 1216-1222
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Isoamyl alcohol oxidase (IAAOD) was purified to apparent homogeneity on SDS-PAGE from ultrafiltration (UF) concentrated sake. IAAOD was a glycoprotein, a monomeric protein with an apparent molecular mass of 73 and 87 kDa, by SDS-PAGE and gel filtration on HPLC, respectively.
      IAAOD showed high substrate specificity toward C5 branched-chain alkyl alcohol (isoamyl alcohol), and no activity toward shorter (C1-C4) or longer (C7-C10) alkyl alcohols tested. IAAOD was stable between pH 3.0-6.0 at 25°C. The optimum pH was 4.5 at 35°C. Heavy metal ions, p-chloromercuribenzoate (PCMB), hydrazine, and hydroxylamine strongly inhibited the enzyme activity, and an anti-oxidant like L-ascorbate did also.
      Isovaleraldehyde was produced markedly in pasteurized sake by adding purified IAAOD, therefore, we concluded that it was the enzyme that causes formation of mureka, an off-flavor of sake, the main component of which is isovaleraldehyde.
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  • Tatsuya HIRANO, Toshitsugu SATO, Kumiko OKAWA, Katsuhiro KANDA, Kaori ...
    1999 Volume 63 Issue 7 Pages 1223-1227
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Lentinus edodes was isolated from a genomic DNA library and cDNA corresponding to this gene was isolated from a mycelium cDNA library. The L. edodes GPD gene was found to encode a 337-aa protein. By comparison of the cDNA and genomic DNA sequences, the presence of eight introns in the GPD gene was confirmed. The putative amino acid sequence of the L. edodes GPD gene product showed high similarity to those of other basidiomycetes. The results of Southern blot analyses suggested that only one copy of the GPD gene is present in the genome of L. edodes. The promoter region was found to contain a CT-rich stretch, two CAAT boxes and a consensus TATA box. In addition, the transcript of the GPD gene was found to be expressed constitutively and strongly. These results suggest that the promoter of the L. edodes GPD gene may be very useful as a component of transformation vectors.
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  • Toshiyuki MATSUNAGA, Shigeyuki USUI, Shigeo UKAI, Tadashi KIHO, Kazuyu ...
    1999 Volume 63 Issue 7 Pages 1228-1237
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      An endothelial cell growth-suppressing factor (EGSF) was purified from the serum-free conditioned medium of the mouse P388D1 culture in the presence of carboxymethylated curdlan. The purified EGSF showed two bands corresponding to the molecular masses of 55 and 63 kDa by silver staining on a SDS-polyacrylamide gel under reducing conditions. This factor strongly suppressed the proliferation of endothelial cells from bovine artery, human umbilical vein, and human dermal vas capillare and this suppression was observed to be reversible. We found that EGSF was a potent chemoattractant for macrophages and neutrophils. EGSF mediated the adhesion of neutrophils to BAEs and transendothelial migration of neutrophils. Macrophages stimulated by EGSF produced nitrite in a dose-dependent manner. EGSF did not affect the proliferation of T lymphocytes. These findings suggest that EGSF acts not only as a potent inhibitor for the growth of endothelial cells but also an activator for macrophages and neutrophils. Thus EGSF plays a role in an inflammatory response in the endothelium.
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  • Haruna KOURIKI-NAGATOMO, Tomomitsu HATAKEYAMA, Masood JELOKHANI-NIARAK ...
    1999 Volume 63 Issue 7 Pages 1279-1284
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      The pore-forming activity of CEL-III, a Gal/GalNAc specific lectin from the Holothuroidea Cucumaria echinata, was examined using artificial lipid membranes as a model system of erythrocyte membrane. The carboxyfluorescein (CF)-leakage studies clearly indicated that CEL-III induced the formation of pores in the dipalmitoyl phosphatidyl choline (DPPC)-lactosyl ceramide (LacCer) liposomes effectively but not in the DPPC-glucosyl ceramide (GlcCer) liposomes or DPPC liposomes. Such a leakage of CF was strongly inhibited by lactose, a potent inhibitor of CEL-III, suggesting that the leakage is mediated through the specific binding of CEL-III to the carbohydrate chains on the surface of the liposomes. The leakage of CF from the DPPC-lactosyl ceramide liposomes was pH-dependent, and it increased with increasing pH. The immunoblotting analysis and circular dichroism data indicated that upon interaction with liposomes, CEL-III associated to form an oligomer concomitantly with a marked conformational change. Furthermore, channel measurements showed that CEL-III has an ability to form small ion channels in the planar lipid bilayers consisting of diphytanoylphosphatidylcholine and human globoside (Gb4Cer)/LacCer.
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  • Eizo TATSUMI, Daisuke YOSHIMATSU, Masaaki HIROSE
    1999 Volume 63 Issue 7 Pages 1285-1290
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Ovalbumin assumes a highly ordered molten-globule conformation at pH 2.2. To investigate whether or not such structural nature is related to the existence of an intrachain native disulfide bond, the structural characteristics of disulfide-reduced ovalbumin at the acidic pH were compared with those of the native disulfide-intact protein by a variety of analytical approaches. The disulfide-reduced protein was found to assume a partially denatured molten globule-like conformation similar to the disulfide-intact counterpart as analyzed by the CD and intrinsic tryptophan fluorescence spectra and by the binding of a hydrophobic probe of anilino-1-naphthalene-8-sulfonate. The results from size-exclusion chromatography also showed that the disulfide-reduced and disulfide-intact proteins have essentially the same compact, native-like hydrodynamic volume. The disulfide-reduced protein was, however, highly sensitive to proteolysis by pepsin at the acidic pH under the proteolytic conditions in which the disulfide-intact protein was almost completely resistant. Furthermore, on a differential scanning calorimeter analysis the disulfide-reduced protein had an endothermic transition at a much lower temperature (Tm=48.5°C) than the disulfide-intact protein (Tm=57.2°C). Taken together, we concluded that the intrachain disulfide bond should not be directly related to the highly ordered molten-globule conformation of ovalbumin, but that its conformational stability depends on the presence of the disulfide bond.
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Biochemistry & Molecular Biology Notes
  • Eijiro HONJO, Keiichi WATANABE
    1999 Volume 63 Issue 7 Pages 1291-1294
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E. coli cells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E. coli at 25°C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E. coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.
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  • Tohru MINAMINO, Hiroyuki DOI, Kazuhiro KUTSUKAKE
    1999 Volume 63 Issue 7 Pages 1301-1303
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.
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  • Tomomitsu HATAKEYAMA, Tetsuji KAMINE, Yuko KONISHI, Hiromiki KUWAHARA, ...
    1999 Volume 63 Issue 7 Pages 1312-1314
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      A lectin-cationic peptide conjugate, 43-CEL-I, was prepared from an invertebrate C-type lectin, CEL-I, and an amphiphilic α-helical peptide, 43-βAla2 [Ac-(Leu-Ala-Arg-Leu)3-βAla2]. When 43-CEL-I was incubated with rabbit erythrocytes, hemolysis was observed, especially at basic pH. Inhibition experiment using some carbohydrates suggested that hemolytic activity of 43-CEL-I was caused by the interaction between 43-βAla2 portion in the conjugate and the lipid bilayer after binding to the carbohydrate chains on the cell surface by the lectin activity of CEL-I.
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Food & Nutrition Science Regular Papers
  • Michihiro FUKUSHIMA, Sumie DOI, Tetsu OHASHI, Tsuyoshi ENDO, Hidetoshi ...
    1999 Volume 63 Issue 7 Pages 1160-1164
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      The effects of a mixture of organisms on cecal fermentation and cholesterol metabolism in sham-operated and cecectomized rats were investigated. Male F344 rats, allocated into four groups: cecectomized rats fed a mixture of organisms (CEMO), cecectomized rats fed rice bran (CERB), sham-operated rats fed a mixture of organisms (SHMO), and sham-operated rats fed rice bran (SHRB) for 4 weeks. The diets had 0.5% cholesterol and 0.125% sodium cholate added. There were no significant differences in the body weight gain and food intake among the groups. The cecal pH in the SHMO group was significantly lower than that in the other groups. The total cholesterol and (VLDL+IDL+LDL)-cholesterol concentrations in serum were significantly lower in the SHMO group than that in the SHRB group, and the triacylglycerol concentration in the sham-operated rats tended to decrease compared to the cecectomized rats. The fecal cholesterol excretion in the CERB group was higher than that in the other groups, and that in the SHMO group was significantly higher than in the SHRB group. The acetic acid, propionic acid, n-butyric acid, and total short-chain fatty acid concentrations in the cecum contents were significantly higher in the SHMO group than those in the other groups. Streptococcus, Bifidobacterium, and Lactobacillus in the SHMO group tended to be higher than the other groups and Bacteroidaceae in the CEMO and CERB groups were significantly higher than that in the SHMO group. The results demonstrate that the mixture of organisms was fermented with the cecal contents and that the metabolites such as short-chain fatty acid lowered the serum total cholesterol and liver cholesterol concentrations in the rats fed a cholesterol-containing diet.
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  • Hiromi UMETSU, Noriyo IKEDA, Van Chuyen NGUYEN
    1999 Volume 63 Issue 7 Pages 1181-1186
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      This study investigated the effects of Maillard reaction products (MRPs) on the oxidative cleavage and polymerization of BSA (bovine serum albumin) in an aqueous system. In L-ascorbic acid (AsA) and Cu(II) or Fe(III) reaction system, 50-60% of BSA was cleaved under physiological conditions (37°C, pH 7.2). The oxidative cleavage induced by AsA-Cu(II) system was suppressed to the extent of 32-86% by model melanoidins or brown pigments from amino acids and foodstuffs. In the AsA-Fe(III) system, the oxidative cleavage was inhibited to the extent of 45-93% by melanoidins and brown pigments. However, this cleavage was promoted by amino acid Amadori rearrangement products and brown pigment from soy paste. Therefore, MRPs show both suppression and promotion activity on oxidative cleavage of BSA in the system of AsA and a transition metal. The quantity of Amadori rearrangement moiety (ARM) in melanoidins from Lysine and brown pigments molecules from foods was also measured. From these data, it was estimated that the suppression and/or promotion of oxidative cleavage of BSA did not only depend on the quantity of ARM, but also depended on the chemical structure of ARM in melanoidins or brown pigments.
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  • Tohru HIRA, Hiroshi HARA, Yoritaka AOYAMA
    1999 Volume 63 Issue 7 Pages 1192-1196
    Published: 1999
    Released: March 12, 2005
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      We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 μg/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 μg/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.
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  • Jun KAYASHITA, Iwao SHIMAOKA, Misao NAKAJOH, Masato KONDOH, Kunioki HA ...
    1999 Volume 63 Issue 7 Pages 1242-1245
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Growing rats were examined for the influence of a buckwheat protein diet on muscle weight and protein. In experiment 1, the rats were fed on a diet containing either casein or a buckwheat protein extract (BWPE) as the protein source (10%, 20% or 30%) for 5 wk. The relative weights (g per kg of body wt) of the gastrocnemius, plantaris and soleus muscles were higher in the BWPE-fed animals than in the casein-fed ones, but were unaffected by the dietary level of protein. These differences were not associated with growth. In experiment 2, the rats were fed on either a casein or BWPE diet at the 20% protein level for 5 wk. BWPE intake significantly elevated the gastrocnemius muscle weight, carcass protein and water, and reduced carcass fat. These results demonstrate that BWPE consumption causes muscle hypertrophy, elevates carcass protein and water, and reduces body fat.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Tomoaki DEGUCHI, Riichiro OHBA, Seinosuke UEDA
    1999 Volume 63 Issue 7 Pages 1151-1155
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      A purple pigment called hordeumin, a type of anthocyanin-tannin pigment, is produced from barley barn-fermented broth. Hordeumin is a precipitate formed by storing fermented filtrate containing hordeumin precursors in an oxidative environment. Hordeumin formation was inhibited by addition of radical scavengers or antioxidants to the barley bran-fermented filtrate. The addition of iron ion accelerated hordeumin formation, and the addition of DETAPAC, an iron ion chelating reagent, influenced the color tone of hordeumin. These findings suggest that hordeumin formation may be an oxygen-mediated reaction, involving reactive oxygen species. Furthermore, hordeumin formation was accelerated by increasing the hordeumin formation temperature (from 5 to 60°C). The color quality of the hordeumin that was produced at high temperature was almost the same as that hordeumin produced at 5°C.
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  • Mariappan PREMANATHAN, Kandasamy KATHIRESAN, Naoki YAMAMOTO, Hideki NA ...
    1999 Volume 63 Issue 7 Pages 1187-1191
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      A polysaccharide was extracted with 1% sodium carbonate from the bark of Rhizophora mucronata and its antiviral activities against human immunodeficiency virus (HIV) were assessed by an in vitro cell culture system. The anti-HIV activity of the alkaline extract was mainly recovered in the 25-75% ethanol-precipitated fraction. Rhizophora mucronata polysaccharide (RMP) protected MT-4 cells from the HIV-induced cytopathogenicity and blocked the expression of HIV antigens. RMP completely inhibited the viral binding to the cell and the formation of syncytium upon cocultivation of MOLT-4/HIV-1IIIB cells and MOLT-4 cells. These results suggest that RMP inhibited early steps of the virus life cycle especially virus adsorption to the cell.
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  • Karl ZIEGELBAUER, Gerda GRUSDAT, Andreas SCHADE, Wolfgang PAFFHAUSEN
    1999 Volume 63 Issue 7 Pages 1246-1252
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      We describe a 96-well microtiter plate format assay to detect changes in proton permeability in membranes of the pathogenic yeast, Candida albicans. Candida albicans cells were incubated with the lipophilic ester of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluorescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH value of the vacuole due to proton leakage into the cytoplasm. This was paralleled by an increase in BCECF fluorescence intensity, which could be quantified. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possible applications and limitations of this assay in the field of antifungal drug discovery are discussed.
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  • Yasuhiro MATSUO, Masako KURITA, Jae Kweon PARK, Katsunori TANAKA, Tsuy ...
    1999 Volume 63 Issue 7 Pages 1261-1268
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45°C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetyl-glucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N′-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.
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  • Rikiya TAKAHASHI, Kenji YAMAYOSHI, Naoshi FUJIMOTO, Masaharu SUZUKI
    1999 Volume 63 Issue 7 Pages 1269-1273
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      We tried to produce (S,S)-ethylenediamine-N,N′-disuccinic acid [(S,S)-EDDS] with the aid of microorganisms that can degrade the compound. A reaction mixture consisting of ethylenediamine and fumaric acid was incubated with the bacteria isolated from soil and sludge, and we found that several kinds of bacteria such as Sphingomonas, Brevundimonas, Pseudomonas, and Acidovorax produced (S,S)-EDDS. The optimum pH and the temperature for the production was pH 7.5 and 35°C for Acidovorax sp. TNT149 and the bacterial cells produced 90 mmoles of (S,S)-EDDS from 200 mmoles of ethylenediamine and 350 mmoles of fumaric acid under these reaction condition.
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  • Eiichiro KIMURA, Chizu YAGOSHI, Yoshio KAWAHARA, Tsuyoshi OHSUMI, Tsuy ...
    1999 Volume 63 Issue 7 Pages 1274-1278
    Published: 1999
    Released: March 12, 2005
    JOURNALS FREE ACCESS
      Glutamate overproduction in Corynebacterium glutamicum is induced by Tween 40, biotin-limitation, or sublethal amounts of penicillin. Disruption of the dtsR gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. We report here that overexpression of dtsR inhibits the induction of glutamate overproduction. In contrast, the level of DtsR in the wild type strain was found to decrease in the presence of Tween 40 or limited amounts of biotin. Tween 40, biotin-limitation, or dtsR disruption also reduced the activity of 2-oxoglutarate dehydrogenase complex (ODHC), which is involved in the synthesis of succinate from 2-oxoglutarate. These results indicate that decrease in the level of DtsR or a complex containing DtsR triggers the increased synthesis of glutamate from 2-oxoglutarate by lowering the ODHC activity.
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