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Satoko ISEMURA, Tokuji IKENAKA
1973 Volume 74 Issue 1 Pages
1-10
Published: July 25, 1973
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Glycopeptides were obtained from reduced and carboxymethylated Taka-amylase A [EC 3. 2. 1. 1] by digestion with trypsin and chymotrypsin and purified, and their amino acid sequences were determined. The sequence of a glycopeptide obtained with trypsin, T1, was deduced to be Asn-Glu-Trp-Tyr-Asp-Trp-Val-Gly-Ser-Leu-Val-Ser-Asn (CHO)-Tyr-Ser-Ile-Asp-Gly-Leu-Arg. This satisfies the hypothesis that the asparagine linked to the N-acetylglucosamine of the carbohydrate group is followed by residue X and then by a threonine or serine residue.
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I. The Structures of Glycopeptides Derived from Cuttlefish Skin Collagen
Mamoru ISEMURA, Tokuji IKENAKA, Yoshio MATSUSHIMA
1973 Volume 74 Issue 1 Pages
11-21
Published: July 25, 1973
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The structures of several glycopeptides obtained from cuttlefish skin collagen by Pronase-P digestion were established and compared with those of three known glycopeptides from vertebrates. The results indicate that around the carbohydrate-carrying hydroxylysine residue there exists a common amino acid sequence, Gly-X-Hyl-Gly-Y-Arg, which seems to act as a recognition sign during the glycosylation processes of rnllacrpn molecules.
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Tohoru NAKAMURA, Toyoko OHKAWA, Jiro KOYAMA
1973 Volume 74 Issue 1 Pages
23-32
Published: July 25, 1973
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Immunization with varying amounts of ovalbumin emulsified in Freund's complete adjuvant was studied for its effect on the production of IgGl and IgG2 antibodies in guinea pigs and also on the precipitability of these antibodies with the antigen. Administration of excessive doses of ovalbumin reduced the production of IgGl and IgG2 antibodies. In particular, IgG2 antibody production was markedly suppressed in the groups of animals receiving excess antigen. The precipitability of these two classes of antibody also decreased as the immunizing doses were increased. Both IgGl and IgG2 antibodies produced in the groups given a minute amount of ovalbumin precipitated almost completely. However, antibodies produced in the groups given higher doses of ovalbumin did not precipitate completely. The effect of the immunizing dose on the precipitability was more marked with the IgG2 antibody than the IgG1 antibody.
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Makoto KAWAKAMI, Kensuke SHIMURA
1973 Volume 74 Issue 1 Pages
33-40
Published: July 25, 1973
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Glycine, alanine, and serine tRNAs from the silk gland of silkworm (
Bombyx mori, L.) were fractionated by the combination of three different column chromatographic procedures, namely DEAE-Sephadex A-50 column chromatography, reversed phase column chromatography with a system of dimethyldilaurylammonium chloride and isoamyl acetate and Hyamine-reversed phase column chromatography.
Glycine tRNA was separated into three components of high purity. Alanine tRNA was separated into six components. Among them, four tRNAs were of purities greater than 70%. Serine tRNA was separated into five components and their purities were less than 50%.
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Ikuo MURONE, Kikuo OGATA
1973 Volume 74 Issue 1 Pages
41-48
Published: July 25, 1973
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The distribution of the isozymes of creatine kinase [EC 2. 7. 3. 2] of muscle and brain tissues on starch-gel electrophoresis was investigated by means of the direct extraction method. The intracellular distribution of creatine kinase in these tissues was also studied. The following results were obtained:
1) There were three isozymes of creatine kinase in the 20, 000×
g supernatant of human fetal skeletal muscle and two isozymes in that of adult rabbit muscle. Two isozymes of creatine kinase were present in the 20, 000×
g supernatant of human fetal brain and two isozymes in that of adult rabbit brain, although one of the latter was very unstable and sometimes undetectable.
2) The intracellular distribution of creatine kinase activity was similar in adult rabbit muscle and brain tissues, that is, the cell sap as well as the microsomal fraction had markedly higher activity than the mitochondrial fraction. The nerve ending fraction in brain tissue also had an enzyme activity comparable with that of mitochondria.
3) The creatine kinase of cell sap had one or two isozymes showing similar electro phoretic mobility to those of the microsomal fraction in the brain or muscle tissues of the adult rabbit, although the enzymatic activities of the two subcellular fractions were rather different in the two kinds of tissues. The major isozymes of cell sap and the microsomal fraction of brain tissue showed higher electrophoretic mobility than those of muscle tissue.
4) Rabbit muscle mitochondria had a specific type of isozyme migrating near the origin, while rabbit brain mitochondria had two kinds of creatine kinase which were of supernatant and mitochondrial types on starch-gel electrophoresis.
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Fumito MATSUURA, Toshiko MATSUBARA, Akira HAYASHI
1973 Volume 74 Issue 1 Pages
49-57
Published: July 25, 1973
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Two types of ceramide 2-N-methylaminoethylphosphonates showing different chromatographic behavior on thin layer chromatography were found in the viscera of
Turbo cornutus.
Relatively non-polar ceramide 2-N-methylaminoethylphosphonates were isolated from the viscera of
T. cornutus by solvent extraction and were purified by silicic acid column chromatography and by mild alkaline hydrolysis.
The ceramide 2-N-methylaminoethylphosphonates were degraded into ceramides and 2-N-methylaminoethylphosphonic acid by enzymatic hydrolysis with phospholipase C (
Clostridium welchii) [EC 3. 1. 4. 3]. The ceramides were analyzed by gas-liquid chromatography-mass spectrometry as their trimethylsilyl ether derivatives and the molecular species were identified as N-hexadecanoyl C
16-, C
17-, C
18-, and branched C
18-sphingosine and C
18- and C
22-spingadienine.
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Akira YAMAMOTO, Katsunori ISHIKAWA, Suzue UHARA, Susumu ADACHI, Mitsuo ...
1973 Volume 74 Issue 1 Pages
59-65
Published: July 25, 1973
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The association between lipid components in blood serum in normo- and hyperlipemia was investigated in the present study. There was a close correlation between total cholesterol and sphingomyelin, expressed by the formula: (cholesterol)=a+b (sphingomyelin), with a correlation coefficient of 0.92 for normotriglyceridemia and 0.90 for hypertriglyceridemia. A difference in the slope of the correlation curve (b) between these two groups seems to be due to the presence of cholesteryl ester as a load instead of the original constituent of the lipoprotein.
There was an association between lecithin and triglyceride shown by the formula, (triglyceride)=a•(lecithin excess)
k, where lecithin excess is defined as: (lecithin+lysolecithin-2×sphingomyelin).
The fatty acid composition of lecithin and triglycerides in blood serum was similar to the fatty acid composition of the corresponding lipid fractions in the liver. In the liver and the xanthoma of the skin in cases of hyperlipemia, there was an increase in acidic glycerophospholipid, lysobisphosphatidic acid or bis-(monoacylglyceryl) phosohate.
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Yoshiaki YANO, Tahei NEGI, Masachika IRIE
1973 Volume 74 Issue 1 Pages
67-76
Published: July 25, 1973
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1) Yeast inorganic pyrophosphatase [EC 3. 6. 1. 1, PP
iase] was found to be inactivated by iodoacetamide.
2) The rate of inactivation of PP
iase by iodoacetamide was minimum at pH 7.0 in the range tested.
3) When PP
iase was inactivated by iodoacetamide at pH 5.5 and 8.0, it was found that about one and three carboxamidomethyl groups were incorporated into PPiase, respectively.
4) The ammo acid residue of PPiase modified by this reagent at pH 5.5 was identified as methionine by paper chromatography and paper electrophoresis of the hydrolysate of
14C-iodoacetamide-modified enzyme.
5) The amino acid residues modified by iodoacetamide at pH 8.0 were identified as methionine, lysine, histidine, and cysteine. The molar ratio of these modified amino acids was 1.2, 0.9, 0.7, and 0.2.
6) Fluorescence emission spectrum, spectrophotometric titration of phenolic groups, N-bromosuccinimide (NBS) oxidation of tryptophan residues and solvent perturbation difference spectrum of carboxamidomethylated PP
iases were studied, and it was concluded that the environment of the tryptophan residues in modified PP
iase differs markedly from that in native PP
iase.
7) On the basis of the above experiments, the importance of the methionine residue for the enzymatic activity of PP
iase was deduced and the possible role of the methionine residue in PP
iase activity was also discussed.
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Harold SWAISGOOD, Masato NATAKE
1973 Volume 74 Issue 1 Pages
77-86
Published: July 25, 1973
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The carboxyl groups of bovine liver L-glutamate dehydrogenase [EC 1. 4. 1. 3] were modified to various extents by coupling with glycine methyl ester in the presence of l-ethyl-3-dimethylaminopropylcarbodiimide (EDC). By properly selecting the reaction conditions, the number of carboxyl groups modified per polypeptide chain could be varied from 2-3 to nearly complete modification. In the “native” form, 30-40 fewer carboxyl groups react with EDC than under conditions favoring unfolding and subunit dissociation. Comparison of the number reacting with the theoretical number available (
1, 2) shows that essentially all groups are reactive under the latter conditions. The less reactive groups may participate in tyrosyl-carboxylate hydrogenbonds at the site of subunit interactions (
3, 4). Kinetic studies of the enzymatic oxidation of NADH or reduction of NAD
+ by two modified enzyme preparations indicated a general increase in their apparent Michaelis constants as compared to those of the native enzyme. The modified enzymes, however, showed greater degrees of activation by high glutamate concentrations. Moreover, the modified enzymes lost all of the substrate inhibition effects displayed by the native enzyme for NADH and α-ketoglutarate.
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Naoki YAMANAKA, Masako HAGIHARA, Kunio YAGI
1973 Volume 74 Issue 1 Pages
87-93
Published: July 25, 1973
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1. In the presence of 2mg% of phosphotungstic acid (PTA), the release by AMP of the controlled respiration of rat liver mitochondria was inhibited, while that by ADP was not. Decreasing the concentration of PTA to 1mg%, the release by AMP occurred after a definite lag period.
2. In the presence of 2 mg% of PTA, the formation of ADP from AMP and ATP by intact rat liver mitochondria was inhibited, while AMP and ATP were formed from ADP regardless of the presence of PTA.
3. Incorporation studies using radioactive AMP and ADP indicated that penetration into the mitochondria was markedly inhibited by PTA for AMP but only slightly for ADP, explaining the above results.
4. These results suggest that PTA affects a site between the outer membrane and the adenylate kinase [EC 2. 7. 4. 3] loci in rat liver mitochondria.
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I. Isolation and Characterization of Anomalous Heavier 3O S Ribonucleic Acid
Kazuo YANAGI, Koujiro ISO
1973 Volume 74 Issue 1 Pages
95-102
Published: July 25, 1973
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From purified ribosomes of yeast,
Saccharomyces cerevisiae, harvested in the log phase, an RNA sample containing a component (30 S) heavier than the usual 18 and 26 S ribosomal RNA (rRNA) was obtained by extraction using phenol, bentonite, and EDTA. The amount of the heavier RNA was 20-30% of the rRNA preparation. RNase treatment proved this heavier component to be RNA. This heavier RNA component was isolated as a centrifugally homogeneous component by repeated sucrose gradient centrifugal fractionation. The sedimentation coefficients of rRNA
from yeast,
s20,
w, were 18, 26, and 30 S. Comparison of molecular weights calculated from the experimental equation relating the sedimentation coefficient with molecular weight, and comparative examination of the base composition, of 30, 26, and 18 S rRNA gave results which were compatible with the possibility that 30 S RNA is a complex of 26 and 18 S rRNA. However, it does not seem that this 30 S RNA is formed by association of 26 and 18S rRNA due to the presence of protein or Mg
2+. Since this 30 S RNA is almost devoid of template activity for amino acid incorporation
in vitro, it is not a messenger-like RNA.
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Takeshi SHIGA, Kiyoshi SHIGA
1973 Volume 74 Issue 1 Pages
103-109
Published: July 25, 1973
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Visible absorption and circular dichroism spectra of D-amino-acid oxidase [EC 1. 4. 3. 3] and its complexes with α-keto acids and benzoic acid were analyzed into their component vibronic bands. Each of two π-π
* transition bands was separated into three component bands (bands I-VI). In the presence of organic solvent, all the component bands shifted toward blue and the magnetic moment of the 380mμ transition band diminished, indicating that the chromophore, the isoalloxazine ring system, had come into contact with the solvent. Upon complexing with acids, the component bands of the 450mμ transition showed a slight red shift, while those of the 380mμ transition showed a slight blue shift. The degree of such spectral alterations depended on the structure of the acids and was parallel to the binding strength (in the order: pyruvate<other α-keto acids<benzoate). Organic solvent did not affect the spectra of the complexes; thus the chromophore is probably buried. The phenomena could be related to the conformational change induced by complex formation.
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Kiyoshi SHIGA, Akio ISOMOTO
1973 Volume 74 Issue 1 Pages
111-115
Published: July 25, 1973
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1) The reactions of high and low temperature conformational states of the monomer of D-amino acid oxidase [EC 1. 4. 3. 3] with quasi-substrates (benzoate,
p-aminobenzoate, and
m-nitrobenzoate) and substrates (D-alanine, D-methionine, and DL-proline) were studied at various temperatures (
ca. 38-5°C) by the kinetic method.
2) All the quasi-substrates tested were competitive inhibitors at all temperatures tested.
3) The inhibition constants,
Ki, of the quasi-substrates were obtained by Dixon plots, at various temperatures. The relationships between log
Ki and the reciprocal of absolute temperature were continuous with no temperature break. Therefore, it appears that there are no functional differences in binding with quasi-substrates between monomer in the high and low temperature conformational states of this enzyme. Thermodynamic binding data were calculated.
4) The specific rate constants and Michaelis constants of the overall reaction of this enzyme with D-alanine, D-methionine, and DL-proline were obtained at various temperatures. The relationships between the logarithm of the specific rate constants and Michaelis constants, and the reciprocal of the absolute temperature were continuous and had no temperature break. Therefore, there are no functional differences in the values of the specific rate constants and Michaelis constants between the high and low temperature conformational states of the monomer of D-amino acid oxidase.
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Kunitada SHIMOTOHNO, Kin-ichiro MIURA
1973 Volume 74 Issue 1 Pages
117-125
Published: July 25, 1973
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RNA polymerase associated with cytoplasmic polyhedrosis virus, which contains double-stranded RNA as a genome, synthesizes single-stranded RNA
in vitro. The optimal conditions for RNA synthesis by this enzyme were investigated.
The RNA product has the same size distribution as the genome segments, showing two fractions on glycerol concentration gradient centrifugation. Each fraction of the synthesized RNA hybridized specifically with the corresponding size fraction of denatured genome RNA segments.
Only after transcription has been carried out over the whole length of each genome segment does the single-stranded RNA product leave the virus particle. It seems that the transcription of every segment starts at the same time, although the transcribed RNA for the shorter segment group is released from the virion faster than that for the larger group.
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Hiroki KASEDA, Tomoo NOGUCHI, Ryo KIDO
1973 Volume 74 Issue 1 Pages
127-133
Published: July 25, 1973
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The biosynthetic routes to 2-aminoacetophenone and 2-amino-3-hydroxyacetophenone were studied. The former was produced from kynurenine via kynurenamine and the latter from 3-hydroxykynurenine
via 3-hydroxykynurenamine in rat liver extracts. The decarboxylation of kynurenine to kynurenamine was catalyzed by the 100, 000×
g supernatant fraction of a 0.25
M sucrose homogenate of rat liver, and that of 3-hydroxykynurenine to 3-hydroxykynurenamine by the rat liver mitochondrial fraction. The conversion of kynurenamine and 3-hydroxykynurenamine to the corresponding
aminoacetophenones proceeded nonenzymically.
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Yoshihiro FUKUSHIMA, Yuji TONOMURA
1973 Volume 74 Issue 1 Pages
135-142
Published: July 25, 1973
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The existence of two kinds of high energy phosphorylated intermediate, EP, with and without bound ADP, in the reaction of Na
+-K
+-dependent ATPase [EC 3. 6. 1. 3] was indicated by the following results.
1. Only one type of EP,
i.e., that for which decay is unaffected by the addition of ADP, was observed in the presence of high concentrations of Mg
2+. On adding K
+, a fraction of the EP decayed rapidly, and then the time course of EP decomposition followed first-order kinetics. The initial rapid decay of EP on addition of K
+ was accompanied by the formation of an equal amountof ATP. The amount of ATP formed was equal to that calculated on the basis of the mechanism previously proposed by Kanazawa
et al., that the equilibrium between an enzyme-ATP complex and EP shifts toward formation of the enzyme-ATP complex on adding K
+.
2. When NEM-treated enzyme was used in the presence of low concentrations of Mg
2+, two types of EP were observed. One was sensitive, and the other was insensitive to ADP. The decay of the former type was accelerated by the addition of ADP. The percentage of ADP-sensitive EP was zero in the initial phase of the reaction after adding ATP, and increased with time to a steady-state level.
3. These two experimental results strongly support the modified mechanism for the Na
+-K
+-dependent ATPase reaction proposed by Kanazawa
et al., in which the two types of EP in the ATPase reaction with and without bound ADP are both high energy forms. The results are in disagreement with the mechanism proposed by Fahn, Albers, Post, and co-workers in which high energy and low energy EP intermediates are formed sequentially in the reaction of transport ATPase.
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Seiki KURAMITSU, Kiyoshi IKEDA, Kozo HAMAGUCHI
1973 Volume 74 Issue 1 Pages
143-154
Published: July 25, 1973
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Interactions of methyl 6-deoxy-N-acetyl-α-D-glucosaminide (α-Me-6-deoxy-NAG), acetamides, and alcohols with hen egg-white lysozyme [EC 3. 2. 1. 17] were studied by means of the changes in the tryptophyl circular dichroic (CD) bands at 295 and 305nm. The effects on the CD spectrum of lysozyme and the binding energy of α-Me-6-deoxy-NAG were essentially the same as those of methyl α-D-glucosaminide (α-Me-NAG). This indicates that the hydrogen bond between O (6) of β-NAG, α-Me-NAG, or β-Me-NAG and the indole NH of Trp 62 is not important for the binding of these saccharides. Acetamides as well as alcohols were shown to interact with lysozyme at the substrate-binding subsite C. The binding of alcohols was stronger than that of acetamides for compounds having the same alkyl chain length. Ethanol had the largest binding constant among the alcohols studied. The binding constants of N-2-hydroxyalkylacetamides were larger than those of the corresponding N-alkylacetamides.
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Alexander McPHERSON, Alexander RICH
1973 Volume 74 Issue 1 Pages
155-160
Published: July 25, 1973
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Large rhombohedral crystals of canavalin have been produced through the action of trypsin on a crude aqueous extract of Jack Bean meal. The crystals are of space group R32 with α
hex=136Å and
chex=75Å (α
rhom=82.4Å and γ=111°). Electrophoresis on SDS-polyacrylamide gels reveals only a single band corresponding to a molecular weight of about 19, 500. Since the molecular weight of the molecule in solution has been reported to be 113, 000, we conclude from the crystallographic and electrophoretic results that canavalin is a hexamer composed of six identical subunits each of molecular weight 19, 500 and arranged according to point group symmetry 32.
Metal analysis, though inconclusive, indicates the presence of Mg
2+, Ca
2+, and Zn
2+ in the crystals. Electron micrographs show the crystals to be quite ordered even after dehydration and to show a unique staining pattern.
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Kazuo FUKUSHIMA, Ryo SATO
1973 Volume 74 Issue 1 Pages
161-173
Published: July 25, 1973
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The cytochrome
b5-like hemoprotein associated with the outer membrane of rat liver mitochondria (OM-cytochrome) was found to be more susceptible to solubilization with trypsin [EC 3. 4. 4. 4] than microsomal bound cytochrome
b5. Based on this finding, a condition for tryptic digestion was set up under which OM-cytochrome could be preferentially released from crude outer mitochondrial membrane preparations leaving most, if not all, cytochrome
b5 of contaminating microsornes still attached to the membrane.
The OM-cytochrome thus solubilized was highly purified by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified cytochrome obtained in one experiment (Prep I) was separated into two hemoprotein bands upon polyacrylamide gel electrophoresis, whereas the sample purified in a second experiment (Prep II) gave three hemoprotein bands. Both preparations were, however, free of any other protein contaminants. Evidence was obtained to indicate that the extra band in Prep II was mainly due to cytochrome bs derived from contaminating microsomes. The other two bands of Prep II as well as those of Prep I seemed to correspond to OM-cytochrome. The separation of OM-cytochrome into two bands suggested that the solubilized hemoprotein had suffered certain proteolytic modifications during tryptic digestion.
The molecular weight (about 12, 000) and behavior upon DEAE-cellulose column chromatography of purified OM-cytochrome were closely similar to those of trypsinsolubilized microsomal cytochrome
b5. However, the two cytochromes differed from each other in immunological properties. Small differences were also detected in their absorption spectra. Furthermore, the tryptic peptide map of apo-OM-cytochrome was clearly different from that of apo-cytochrome
b5.
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Koko MURAKAMI, Yoko IMAMURA, Sadahiko ISHIBASHI
1973 Volume 74 Issue 1 Pages
175-177
Published: July 25, 1973
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Kunio YAGI, Nobuhiko SUGIURA, Michiko MIZUNO
1973 Volume 74 Issue 1 Pages
179-181
Published: July 25, 1973
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Kiyozo KAWAI, Hiroshi MIZUSHIMA
1973 Volume 74 Issue 1 Pages
183-185
Published: July 25, 1973
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Hirotomo YAMADA
1973 Volume 74 Issue 1 Pages
187-189
Published: July 25, 1973
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Michihiro SUGANO
1973 Volume 74 Issue 1 Pages
191-193
Published: July 25, 1973
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Shin-ichiro EJIRI, Hideharu TAIRA, Kensuke SHIMURA
1973 Volume 74 Issue 1 Pages
195-197
Published: July 25, 1973
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Tadao TERAO, Toshiaki OSAWA
1973 Volume 74 Issue 1 Pages
199-201
Published: July 25, 1973
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1973 Volume 74 Issue 1 Pages
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1973 Volume 74 Issue 1 Pages
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1973 Volume 74 Issue 1 Pages
e2b-e3b
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