The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 65, Issue 5
Displaying 1-24 of 24 articles from this issue
  • II. Effects of pH and Salts
    TAIJI IMOTO, YASUTAKA DOI, KATSUYA HAYASHI, MASARU FUNATSU
    1969 Volume 65 Issue 5 Pages 667-671
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The effects of pH and salts on the activity of lysozyme [EC 3. 2. 1. 17] and the formation of the enzyme-substrate complex were studied spectrophotometrically and by chromatography on a column of an insoluble substrate, carboxymethyl (CM) chitin. The activity of lysozyme was strongly inhibited by neutral salts at high concentrations. In contrast, the formation of the enzyme-substrate complex measured by difference spectrophotometry and chromatography on CM-chitin column increased on increasing the salt concentration. The effect of salts (ions) on the activity depended upon their ionic radii. These effects of salts could not be explained merely by their influence on the ionization of amino acid side chains of the lysozyme molecule. The effects of salts on the complex formation and the activity, therefore, seemed to be mediated by a fine conformational change at the active site, resulting in an unsuitable arrangement of catalytic groups for the catalytic reaction and a strong interaction between the lysozyme molecule and the substrate.
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  • MITSUO NAKAGAWA, MITSURU UCHIYAMA
    1969 Volume 65 Issue 5 Pages 673-677
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Desaturation in vitro of palmitic acid by a postmitochondrial supernatant of rat liver homogenates proceeded about 3 times faster than that of stearic acid. When mitochondria and carnitine were added to the system 15min after the start of incubation, palmitoleic acid produced from palmitic acid decreased more rapidly than oleic acid from stearic acid. However, the rate of oxidation of exogenous palmitoleic acid by isolated mitochondria was about the same with that of exogenous oleic acid. Incorporation of oleic acid into esterified lipids by the postmitochondrial supernatant was about 2 times faster than that of palmitoleic acid, and the release of palmitoleic acid from esterified lipids seemed to be more rapid than that of oleic acid. These findings suggested that the discrepancy between the efficiency of desaturation of the two saturated acids in vitro and the ratio of the two mono-unsaturated acids found in vivo was due to the difference in the rates of incorporation of the two unsaturated acids into esterified lipids.
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  • TAKAYUKI OZAWA
    1969 Volume 65 Issue 5 Pages 679-691
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. At the point of re-establishment of controlled respiration which was released by the addition of AMP, a significant amount of modified AMP, “AMP”, was found in the mitochondrial suspension.
    2. Distribution of 14C-nucleotides and esterified 32P in and outside of mitochondria proved that there was negligible accumulation of nucleotides by the mitochondria during oxidative phosphorylation.
    3. Studies using 5'-adenylate deaminase showed that the conversion of AMP to “AMP” was a respiration-dependent process. The conversion proceeded in the absence of added phosphate, even though its rate was slow.
    4. “AMP” was provisionally identified to be acyl-adenylate on thin layer chromatography by comparison of its Rf value with that of synthetic reference compounds other than both 5'-AMP and 3', 5'-cyclic AMP.
    5. Parallel to the accumulation of “AMP” during state 3 respiration, there was accumulation of hydroxamate-reacting material. It was concluded that “AMP” might be a high energy ester derivative of AMP, possibly containing a substrate derived material.
    6. The nature of “AMP” was discussed in relation with the transfer of high energy bonds across the mitochondrial membrane.
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  • MICHIKO HIRAMARU, TSUNEKO UCHIDA, FUJIO EGAMI
    1969 Volume 65 Issue 5 Pages 693-700
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. Three RNases were isolated from Physarum polycephalum and designated RNase Pp-1, RNase Pp-2 and RNase Pp-3.
    2. They are thermostable.
    3. The approximate molecular weights estimated by get filtration of RNases Pp-1 and Pp-2 are 40, 000 and that of RNase Pp-3 is 10, 000.
    4. The optimum pH values of RNases Pp-1, Pp-2 and Pp-3 are pH 6.7, pH 4.5 and pH 5.5, respectively.
    5. RNase Pp-1, is a guanyloribonuclease [EC 2. 7. 7. 26].
    6. RNase Pp-2 is a nonspecific RNase [EC 2. 7. 7. 17].
    7. RNase Pp-3 may be classified as a nonspecific RNase [EC 2. 7. 7. 17], but it attacks purine nucleotide bonds preferentially.
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  • MICHIKO HIRAMARU, TSUNEKO UCHIDA, FUJIO EGAMI
    1969 Volume 65 Issue 5 Pages 701-708
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. Two nucleases hydrolyzing both RNA and denatured DNA (Nucleases Pp-1, and Pp-2) and one ribonuclease (RNase Pp-4) were isolated from Physarum polycephalum in addition to the three RNases (RNases Pp-1, Pp-2, and Pp-3, ) reported in the preceding paper (1).
    2. They are thermolabile.
    3. The pH optima of nucleases Pp-1 and Pp-2 are pH 4.5, and that of RNase Pp-4 is pH 4.0.
    4. Nuclease Pp-1 and nuclease Pp-2 hydrolyze denatured DNA as well as RNA, but not native DNA.
    5. Nuclease Pp-1 hydrolyzes RNA forming nucleotides with terminal 3'-phosphate.
    6. Nuclease Pp-2, hydrolyzes RNA to produce nucleotides with terminal 5'-phosphate. It has no base specificity.
    7. RNase Pp-4 hydrolyzes RNA forming nucleotides bearing 5'-phosphate group with purine preference in regards to 3'-terminal nucleotides.
    8. Nuclease Pp-2 and RNase Pp-4 are strongly activated by Zn++
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  • I. Presence of Membrane Subunits and the Physical Properties
    MITSUHIRO YANAGIDA, HARUHIKO NODA
    1969 Volume 65 Issue 5 Pages 709-720
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The isolation method of cell membrane of vegetative myxamoebae of Dictyoslelium discoideum is described.
    The isolated cell membrane was morphologically pure with the thickness of 70-90Å, and composed of 65-70% of dry weight of protein and 25-30% of lipid.
    Isolated cell membrane was dissolved completely into structural units in alkaline pH of above 11 as well as in solutions which contained detergents, urea or acetic acid. The nature of the structural unit was investigated mainly with sedimentation and electron microscopy. Lipid-free protein fraction was also dissolved in 0.1M NaOH.
    The structural unit was a lipoprotein particle of which the molecular weight was about 160, 000 and had a sedimentation coefficient of 7S. This particle seemed to be globular with a diameter of 70-100Å. Lipid-free protein fraction showed a single peak in sedimentation and electrophoresis. The sedimentation coefficient was 3.3S and the molecular weight was about 65, 000.
    An intermediate filamentous structure, which was a linear aggregate of 7S subunits, appeared in the process of degrading the membrane into particles in an alkaline solution.
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  • II. Reassociation of Membrane Subunits
    MITSUHIRO YANAGIDA, HARUHIKO NODA
    1969 Volume 65 Issue 5 Pages 721-739
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Reassociation of 7S lipoprotein subunits and 3.3S protein subunits obtained from the cell membrane of vegetative myxamoebae of Dictyostelium discoideum was investigated with electron microscopy, turbidometry and sedimentation.
    7S subunits reassociate into five kinds of aggregates; i.e., unit membrane structure, thick membrane of about twice as thick as that of the unit membrane, filamentous structure, partial aggregates and random aggregates. The proportion of various aggre-gates depended on the conditions for reassociation. The rate of subunit reassociation, hydrogen ion concentration, temperature, metal ion concentration, the valence of metal ion and the presence of substances such as urea influenced the subunit interaction and the morphology of the product.
    Lipid-free protein subunits of the membrane reassociated into membrane structure by going through stages of intermediate aggregates, filaments and then side-by-side aggregation of filaments.
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  • II. Dependence of the Rates of Formation of Phenol, Phenyl α-Glucoside and Maltotriose on the Substrate Concentration
    HOZUMI YOSHIDA, KEITARO HIROMI, SÔZABURO ONO
    1969 Volume 65 Issue 5 Pages 741-750
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. The hydrolysis of phenyl α-maltoside (φM) catalyzed by crystalline saccharifying α-amylase [EC 3. 2. 1. 1] of Bacillus subtilis was studied at various substrate concentrations at 25°C and pH 5.4 in 0.02M acetate buffer by thin layer chromatography, phenol reagent method and modified Somogyi-Nelson's method. The rates of formation of phenol (φ), vφ, phenyl α-glucoside (φG), vφG, and maltotriose (MT), VMT, were deter-mined, and their dependencies on the substrate concentration were investigated. The effect of pH on the rates of formation of φ and φG was examined at a fixed substrate concentration.
    2. In the parallel reactions forming φ and φG from φM, vφ follows the Michaelis-Menten equation over the whole range of substrate concentration, s (0.12-120mM). On the other hand, the plot of vφG versus substrate concentration shows a sigmoidal curve at lower substrate concentrations (0.12-10mM), and the plot of s2/vφG versus s gives a straight line, although the rate obeys the Michaelis-Menten kinetics at higher substrate concentrations (11-120mM). The dependency of vφG/vφ on s shows a saturation curve of the Michaelis-Menten type.
    3. The optimum pH for the processes forming phenol and phenyl α-glucoside is between 5.3 and 5.4, and the pH values, which give one half the velocity at the opti-mum pH, are 4.0 and 6.75 for formation of phenol, and 3.7 and 7.05 for formation of phenyl α-glucoside.
    4. The dependencies of vφ and vφG on the substrate concentration can be explained neither by a simple mechanism of a single active center producing both phenol and phenyl α-glucoside nor by a mechanism of two active centers each forming a single set of products, phenol and maltose or phenyl α-glucoside and glucose. Besides an active site (active center), an activator site to which the substrate is bound is considered necessary for the formation of φG, while such an additional site is not necessary for the formation of phenol.
    5. The apparent rate of formation of maltotriose, VMT, was found to be propor-tional to the product of vφG and vφ.
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  • SHIGERU NAKAYAMA, SHIZUKO AMAGASE, MOTOKO TAHARA, KEIKO OKUMURA
    1969 Volume 65 Issue 5 Pages 751-754
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    A simple, rapid micro-method is described for identification and differentiation of amylases in a limited quantity of preparation. The method is a modification of the ‘oligosaccharide map’ procedure, involving chromatography of malto-oligosaccharides in one direction on a thin layer of microcrystalline cellulose (Avicel SF), incubation of the oligosaccharides with a small quantity of amylase preparation, separation of the hydrolytic products by chromatography in a second direction and location of the hydro-lytic products.
    Partially purified amylase from the root of Japanese radish (Raphanus sztivus L. var. aconthiformis Makino) was analyzed by this method. β-Amylase [EC 3. 2. 1. 2] seemed to be the main component, with a glucose-forming amylase or glucoamylase [EC 3. 2. 1. 3] as a minor component.
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  • ATSUSHI SAKURAI, MIKI GOTO
    1969 Volume 65 Issue 5 Pages 755-757
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
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    7-Amino-2-(1', 2'-dihydroxyäthyl)-5-hydroxy-3-methylthiothiothieno[3, 2-g]pteridin wurde synthetisiert und die Identität der synthetisierten Substanz mit dem Urothion festgestellt.
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  • TATSUYA SAMEJIMA, MASAKO KITA
    1969 Volume 65 Issue 5 Pages 759-766
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Optical rotatory dispersion and circular dichroism of solutions of horse ferri-myo-globin and its derivatives (ferro-, carbonyl-, oxy-, cyano-, azide-, apo- and reconstituted myoglobin) were measured over a wavelength range of 650 to 185mμ. All myoglobins except apo-myoglobin showed distinct positive Cotton effects and circular dichroic bands in the region of Soret absorptions. However, the crossover points of the Cotton effects were essentially identical with those of the positive maxima of the circular dichroic bands and the absorption maxima of the Soret bands. The magnitude of the Soret Cotton effect and circular dichroic band of carbonylmyoglobin was most prominent. In the near-ultraviolet region between 260-280mμ all proteins showed positive circular dichroic bands, which may be due to asymmetry of the aromatic amino acid groups. The intensities of these bands were enhanced giving a blue shift when the myoglobin was complexed with the ligand molecules. In the ultraviolet region intense negative circular dichroic bands near 222mμ and 207mμ were present in accord with the cor-responding Cotton effects having a trough at 233mμ and a peak near 200mμ. Helix contents, based on the 233mμ trough and the band intensity at 222mμ, were estimated for all the proteins. Within experimental error, horse ferrimyoglobin and its deriva-tives have almost the same helix content of about 70%, whereas apomyoglobin has less helix content, approximately 50%. This indicates that the addition of ligands to the heme group causes no drastic conformational change in the protein moiety, while the removal of heme has an effect on the diminution of the helix content. Reconstituted ferrimyoglobin regained essentially the same conformation as the native protein.
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  • FUMIO SAWADA
    1969 Volume 65 Issue 5 Pages 767-776
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Bovine pancreatic ribonuclease [EC 2. 7. 7. 16] (RNase) was inactivated by irradia-tion with ultraviolet light of 330-365mμ in the presence of a substrate analogue, 4-thiouridine-2'(3')-phosphate (4-thiouridylic acid), while a little or no inactivation was found in absence of the nucleotide and in the presence of a nucleoside, 4-thiouridine. The inactivation was inhibited by an excess amount of uridylic acid. The inactivation was notable at pH 5.6 and was feeble at pH's 3.6 and 7.6, and the degree of the inactivation was in paralell with the degree of the interaction between the enzyme and the nucleotide. It was deduced from these results that the complexing between RNase and 4-thiouridylic acid is an essential step for the photosensitized inactivation of the enzyme. Oxygen was required for the inactivation.
    A photoproduct of RNase irradiated in the presence of 4-thiouridylic acid was isolated. Chemical analyses of this product revealed that histidyl, phenylalanyl and valyl residues at the active site were intact after the irradiation, but one tyrosyl residue was modified. The spectrophotometric titration of the irradiated protein suggested that the native conformation was retained.
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  • I. Covalent and Non-Covalent Binding of Fluorescein-Isothiocyanate and Fluorescein to Proteins
    HIROSHI MAEDA, NAKAO ISHIDA, HIROSHI KAWAUCHI, KATURA TUZIMURA
    1969 Volume 65 Issue 5 Pages 777-783
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. The reaction of fluorescein-isothiocyanate (FITC) with proteins was studied under various conditions.
    2. It was found that the α-amino groups of the N-terminal amino acids were the prime target of the reaction (at pH<9.5) of FITC with neocarzinostatin and insulin although the ε-amino groups became reactive at higher pH.
    3. Egg white lysozyme [EC 3. 2. 1. 17] and bovine serum albumin were found to bind with FITC even at low pH, while ovomucoid and neocarzinostatin did not bind below pH 7.
    4. Trifluoroacetic acid treatment of fluorescein-thiocarbamylated insulin (FTC-insulin) resulted in the release of terminal FTH-amino acids (Phe and Gly) only. These FTH-amino acids were identified for the first time.
    5. Non-covalent binding of this fluorochrome to proteins was studied with fluoro-scein. The results indicated non-covalent dye-binding of the fluorescein with albumin and Iysozyme [EC 3. 2. 1. 17].
    6. Above results showed that FITC is a useful reagent but that non-covalent dye-binding should be born in mind when used with some proteins.
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  • KEIJI NAKAMURA, AYAKO MATSUSHIMA
    1969 Volume 65 Issue 5 Pages 785-792
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
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    The states of amino acid residues in porcine and bovine trypsins were compared by spectrophotometric titration of tyrosine residues and by using several reagents: β-naphthoquinone-4, 6-disulfonic acid (NQDS) and monochlorotrifluoroquinone (CFQ) for amino groups, H2O2-dioxane for tryptophan, glyoxal for arginine, diazo-l-H-tetra-zole (DHT) for histidine and tyrosine, and cyanuric fluoride for tyrosine. Almost all the amino groups in these trypsins [EC 3. 4. 4. 4] were modified uniformly with NQDS and 5-6 amino groups of bovine trypsin and 4-5 amino groups of porcine trypsin were modified with CFQ. Glyoxal modified two of the total four arginine residues in porcine trypsin and one of the two arginine residues in bovine trypsin. Three of the four histidine residues in porcine trypsin and two of the three histidine residues in bovine trypsin were bisazotized with DHT. Three of the four tryptophan residues in bovine trypsin and all the four tryptophan residues in porcine trypsin were oxidized with H2O2-dioxane. Of the eight tyrosine residues in porcine trypsin three residues had values of pK=10.2, m (the order of the sigmoid ionization curve)=1.0, three values of pK=10.5, m=1.0 and two of pK=12.2, m=3.0. The three residues with a pK value of 10.2 were further classified in terms of their reactivity with CyF into two more reactive and one less reactive residues. The two residues with the highest pK value of 12.2 were not bisazotized while the other six residues were completely bisazotized. These data were compared with the previous observations that the ten tyrosine residues in bovine trypsin were of two types: six which ionized rapidly and reacted with CyF and four which ionized slowly and did not react with CyF. The difference in stability between bovine and porcine trypsins is discussed on the basis of these data, and the participation of a serine residue in the activity of porcine trypsin is postulated.
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  • HARUKI YAMAGUCHI, TOKUJI IKENAKA, YOSHIO MATSUSHIMA
    1969 Volume 65 Issue 5 Pages 793-798
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The homogeneity of a glycopeptide obtained from Taka-amylase A [EC 5. 2. 1. 1], was reinvestigated by column chromatography on Dowcx 50×2 and high voltage paper electrophoresis. About 90 per cent of the glycopeptide was found to be homogeneous and to consist of one mole each of aspartic acid, ser ne and ammonia, 2 moles of N-acetylglucosamine and 6 moles of mannose.
    Sequential periodate oxidation of the purified homogeneous glycopcptide indicated that its carbohydrate sequence is Man-Man-G1eNAc-G1cNAc-NH.
    _??_
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  • YASUO KAGAWA, TOSHIKO TAKAOKA, HAJIM KATSUTA
    1969 Volume 65 Issue 5 Pages 799-808
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    1. Mitochondrial lipids of mouse fibroblasts of the substrain Lp3, one of the sub-lines of L-929 which had been cultured in the lipid-free and protein-free synthetic medium DM-120 for 9 years, were analyzed.
    2. Essential fatty acids were not detected but monounsaturated fatty acids were found to have increased in the mitochondria. The presence of coenzyme Q, inositol, and vaccenic acid was confirmed.
    3. By the addition of linoleic acid into DM-120, considerable changes were induced in the fatty acid composition of each phospholipid fraction, whereas the proportional ratios among phospholipids were little influenced.
    4. Mitochondrial functions, such as oxidative phosphorylation in situ, ATPase [EC 3. 6. 1. 3] activity and electron transport by cytochromes, were kept unchanged in the lipid-free synthetic medium. The oxidative phosphorylation in the isolated mitochon-dria, however, was found unstable, suggesting that it awaits further investigation.
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  • TADASHI INAGAMI, ABRAHAM PATCHORNIK, SHELDON S. YORK
    1969 Volume 65 Issue 5 Pages 809-819
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Derivatives of N-acetyl-L-tyrosine anilide with substituents, p-CH3, p-CH3O, m-CH3O, p-Cl and m-G, on the aniline ring, were synthesized as substrates of chymotrypsin [EC 3. 4. 4. 5]. It was confirmed that the acylation of the enzyme was the rate limiting step for these anilide substrates. It was, therefore, possible to study the electronic effect of the substituents on the catalysis of the acylation. The ρ-σ- plot indicated participation of an acidic group of chymotrypsin in the proton transfer to the substrate molecule. The deuterium isotope effect was observed to depend on the σ- value of the substituent. Such dependence was found to be compatible with a mechanism in which the proton transfer occurs rapidly before the rate limiting step, but not with a mechanism in which the proton transfer occurs simultaneously with the acylation. The pH profile of the catalysis was studied. The temperature dependence of the rate constant of catalysis revealed that the heat of activation changes with the σ- value more steeply than the free energy of activation. This steeper change is compensated for by a change in the entropy of activation in the reverse direction.
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  • MASAHIRO IWAMOTO, YASHUSHI ABIKO
    1969 Volume 65 Issue 5 Pages 821-823
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • KYOKO TORII, YASUYUKI OGURA
    1969 Volume 65 Issue 5 Pages 825-827
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • YAKAKO TOMITA, TATUO OZAWA, ISAO TOMITA
    1969 Volume 65 Issue 5 Pages 829-830
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • KOICHI SUZUKI, TOSHIO ANDO
    1969 Volume 65 Issue 5 Pages 831-834
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • AKIRA KOTAKI, MAKOTO NAOI, MINORU HARADA, KUNIO YACI
    1969 Volume 65 Issue 5 Pages 835-837
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • KEN KADOTA, TOKU KANASEKI
    1969 Volume 65 Issue 5 Pages 839-842
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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  • HAJIME HIRATA, SAKUZO FUKUI, SHINJI ISHIKAWA
    1969 Volume 65 Issue 5 Pages 843-847
    Published: May 25, 1969
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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