CO6-1

D-serine Acting as Competitive Inhibitor on Tryptophanase

A. Shimada1, I. Otsuka1, N. Fujii2 and T. Saito2

ice to stop reaction. 200 μL of the reaction mixture wasfiltered through Amicon Ultra Centrifugal Filters cutting
more than MW 3000, immediately injected on a
Crownpack CR (+) Resolution HPLC resolution column
equilibrated with the degassed eluent through a Degasser, and eluted at a flow rate of 0.9 mL/min at room
temperature with an HPLC pump. L-Trp synthesis from
L-Ser was determined measuring with a CD-1595 detector. Ellipticity [θ] was monitored at λ = 230 nm with
the CD detector, represented in units of mdeg. Ellipticity of the eluent was set up to zero at a wavelength of λ =230 nm, so the peaks of L-Trp appeared on the positive
side.
RESULTS AND DISCUSSION: For kinetic analysis,
TPase activity on L-Trp synthesis was assayed in triplicate at the same substrate concentration, averaged. Lineweaver-Burk plots were drawn based on the leastsquares method. The reciprocal of rate constant (ordinate) versus the reciprocal of substrate concentration(abscissa) was plotted for each concentration of D-Ser,
depicting a typical competitive inhibition plot. Kinetic
constants were calculated from these double-reciprocalplots, and also an inhibition constant KI of D-Ser wasdetermined from their slopes. Km was 44 mM, Vm 2.2
×10-4 mM/s, kcat 0.48/s and KI 419 mM. D-Ser is a competitive inhibitor against TPase in L-Trp synthesis reaction from L-Ser and indole. Competitive inhibitor usuallyshares a highly structural resemblance with its substrate.
D-Ser stereostructure fits into the active site structure of
TPase, promptly binding there. Although D-Ser can bindwith the active site, D-Ser is not involved in L-Trp synthetic reaction. Perhaps D-Ser cannot form aldimine-bond with PLP at the first step of L-Trp synthesis.
However, our previous studies have reported that TPase
becomes active on D-Ser in the presence of triammonium phosphate (TAP) to make the enantioselectivity flexible. TAP is the most important among ammonium
phosphates because it gives TPase the highest activity on
D-Ser. The presence of TAP promotes a small conformational change to TPase to fit D-Ser partly into the catalytic site of the active site and help D-Ser to form theexternal aldimine bond with PLP. This subtle steric interaction, whose detail is still being investigated, are very
necessary to transform D-Ser from inhibitor into active
substrate. Future research will determine a metabolic
pathway of L-Trp synthesis from D-Ser, and also
demonstrate the formation of an aldimine bond between
amino group of D-Ser and PLP at the first step of thisreaction in the presence of TAP.

1

Sustainable Environmental Studies, Graduate School ofLife and Environment Sciences, University of Tsukuba
2
Research Reactor Institute, Kyoto University
INTRODUCTION: A historical riddle about chiralhomogeneity, the origin of L-dominant amino acids inbiological world, has intrigued scientists for many years,
though there has been no general consensus as yet to it.
Life could not been born without homochirality, that is
to say, no homochirality – no life. However, we cannot
imagine at all when and how the homochirality wasestablished on early earth. Amino acids are stereochemically too labile to retain their homochiral state in natural environment because racemization pressure is overwhelmingly strong, especially in aqueous solution. An
elaborate mechanism is necessary to put biologicalworld into homochirally-stable state. The homochiralityof contemporary biological world is fully guaranteed byenzyme enantioselectivity. Since the enzyme enantioselectivity has remained unclear, a deeper understandingof its mechanism is required to unravel all the riddle ofthe origin of homochirality. We have so far researched amechanism of tryptophanase (TPase) enantioselectivity,with the aim of answering this difficult problem. TPasedegrades L-tryptophan (L-Trp) by β-elimination reaction, and also synthesizes L-Trp from L-serine (L-Ser)and indole by β-replacement reaction by use of its absolute enantioselectivity. On the other hand, it is perfectly inert to D-tryptophan (D-Trp) and D-serine(D-Ser) at all. Additionally these D-amino acids inhibit
TPase in L-Trp degradation and L-Trp synthesis from
L-Ser. The present report describes to focus simply on
D-Ser acting on TPase as a competitive inhibitor.
EXPERIMENTAL: Generally speaking, D-amino acidworks as inactive or inhibitor against enzyme. D-Ser
completely has no reaction to TPase. When TPase synthesizes L-Trp from L-Ser and indole, D-Ser inhibits thesynthetic reaction. Since kinetic analysis was effective
to probe the interaction between D-Ser and TPase, we
analyzed D-Ser behavior against TPase in L-Trp synthesis in terms of it. For kinetics, reaction was conducted at 50 °C for 4 hr in Dry Thermo Unit DTU-1B with 0,0.1, 0.2, 0.3, 0.6, or 1 M of D-Ser added in reaction
mixtures that were prepared by combining the required
L-Ser (10-100 mM) with fixed apotryptophanase(apoTPase, 0.46 μM), indole (5.4 mM) and pyridoxal
5'-phosphate (PLP) concentrations (0.4 mM) in 500 μL
of 0.1 M potassium phosphate buffer (PB), adjusted to a
pH of 8.3. After 4hr, the reaction mixture was cool on

採択課題番号 26012 ガンマー線照射された酵素の立体構造変化による立体選択性への影響 通常採択
（筑波大・生命環境系）島田秋彦、大塚郁美（京大・原子炉) 藤井紀子、齋藤 毅
126CO6-2

Study of Localization Estimation of Abasic Sites in DNA Irradiated with
Ionizing Radiation

K. Akamatsu, N. Shikazono and T. Saito1

microliters of water was added to the residue. The fluorescence intensities were measured both at 449 nm (ex.
347 nm for AF350) and at 520 nm (ex. 460 nm for
AF488). After the measurement, the enzyme cocktail
containing DNase I and phosphodiesterase I was added
to the solution, followed by being incubated for 3 h at
37°C. E values were calculated from the donor intensity
before/after the digestion.

Irradiated Cell Analysis Research Group, Quantum Beam
Science Research Center, Japan Atomic Energy Agency(JAEA)
1
Research Reactor Institute, Kyoto University
INTRODUCTION:
DNA lesions induced by ionizing radiation andchemicals can cause mutation and carcinogenesis. In
particular, “clustered damage” site, that is a DNA region
with multiple lesions within one or two helical turns, isbelieved to hardly be repaired. This damage is considered
to be induced, e.g., around high-LET ionizing radiation
tracks. However, detail of the damage is not known. We
have already developed a method for estimating degreeof localization of abasic sites (APs) in DNA using Förster
resonance energy transfer (FRET). The FRET efficiency(E) was calculated using the donor fluorescence intensities before/after enzymatic digestion of the labeled
AP-DNA [1]. First, we have applied the method to 4He2+(2.0 MeV/u, LET: ~70 keV/m)- 12C5+ (0.37 MeV/u,
LET: ~760 keV/m)- and 60Co -irradiated DNA in thesolid state. The results showed that 12C5+ beam likely
produced close APs within a track. The apparent distancecalculated from the E value was approximately 17 basepairs [2]. This finding indicates that direct radiation effect of 12C5+ beam near the Bragg peak produces clustered DNA damage. We have recently applied the method
to DNA in a cell-mimetic radical scavenging condition[3,4]. Here, we show new results using 4He2+ beam and60
Co -rays.

RESULTS AND DISCUSSION:

Fig. 1. Relationship between AP average density and FRET
efficiency for He ion beam (●) and 60Co -rays (○). The solidline indicates a theoretical curve for randomly-distributed APs
in DNA.

As shown in Fig.1, there was no difference in
FRET efficiency (E) between the two radiation sources.
But interestingly, all data points were above the curvefor random distribution. This finding suggests that AP
distribution for the -rays as well as that for the He
beam is not completely random. This tendency is similar to the result using dry DNA as sample for irradiation[2]. A radiation “spur” on or nearby DNA might sometimes produces clustered damage. In any case,knowledge of AP distribution for 60Co -rays as a reference is quite important to study DNA damage by ionizing radiation. More data points and discussions will beneeded to elucidate the problem.

EXPERIMENTS:●Sample preparation and He beam irradiation
The plasmid DNA digested by Sma I was used (linear
form). The DNA was dissolved to be 0.1 g/L in 0.2 M
Tris-HCl buffer (pH 7.5) which is a cell-mimetic condition in relation to radical scavenging capacity. One hundred microliters of the DNA solution was transferred toan irradiation folder [2], and was irradiated with the4
He2+ beam (12.5 MeV/u, LET: ~19 keV/m; TIARA,
JAEA), and 60Co -rays (LET: ~0.2 keV/m; Kyoto
University Research Reactor Institute: KURRI) were also
used as a standard radiation source.

REFERENCES:[1] K. Akamatsu, N. Shikazono, Anal. Biochem. 433(2013) 171-180.
[2] K. Akamatsu, N. Shikazono, and T. Saito, Radiat.
Res. 183 (2015) 105-113.
[3] K. Akamatsu, N. Shikazono, and T. Saito, KURRI
Progress Report 2012 (2012) 256.
[4] K. Akamatsu, N. Shikazono, and T. Saito, KURRI
Progress Report 2012 (2013) 263.

●Preparation of fluorophore-labeled irradiated DNA andthe FRET observation
The irradiated DNA (10 L in water) and 10 L of
100 mM Tris-HCl (pH7.5) were mixed in a microtube.
Two microliters of a mixture containing AF350 (donor
fluorescent probe) and AF488 (acceptor one) with a given molar ratio was added to the DNA solution and was
incubated for 24 h at 35°C. The fluorophore-labeled
DNA was purified by precipitation by ethanol. Twenty

採択課題番号 26017 放射線誘発ＤＮＡ損傷スペクトルの線質依存性に関する研究（原子力機構・量子ビーム）赤松 憲、鹿園直哉（京大・原子炉）齊藤 毅
127

通常採択
共同通常

CO6-3The Effect of Boron Neutron Capture Therapy (BNCT) on
Anti-tumor Immune Response

Suzuki T.1, Horie K.1, Ogawa S.1, Wakamatsu E.1,
Abe R.1, Sakurai Y.2, and Ono K.2

Notably, unlike X ray irradiation, BNCT did not enhance anti-tumor immune response. Although additional immunization with peptide vaccination and CTLA4
blockade after BNCT lead to the induction of immune
response, this combination therapy could not achievetumor regression (Fig.2B and 2C). Our further interest
is why BNCT could not enhance anti-tumor immune response. To understand the anti-tumor immune response
during radiation therapy determines the effective combination with radiation therapy and immune therapy.

1

Recerch Institute of Biomedical Sciences,
Tokyo University of Science2
Research Reactor Institute, Kyoto University
INTRODUCTION: Boron neutron capture therapy(BNCT) is an attractive therapy for local tumor control in
the treatment of brain tumor, melanoma, and so on [1,2].
However, some important issues are remained: the tumor-specific accumulation of highly concentrated boron,
the real-time quantification of boron concentration in
local tumor tissues, and the prevention of re-growth andmetastasis of residual tumor cells after BNCT.
Recently, it was reported that some cancer immune therapies could achieved significant regression of tumor inclinical trials. To achieve successful tumor regression,in this study, we try to establish the combination treatments with BNCT and immunotherapy [3,4].
Results and Discussions:
The effect of BNCT on immune system
Using mouse model, we examined the effect of BNCTon the antigen specific immunological response.
C57BL/6 mice, which were subcutaneously administrated
with 250 mg/kg BPA, and then treated with thermal neutron irradiation (1 MW, 50min in the heavy water facilityof KUR). To assess the immune response, mice were
immunized with antigenson footpad, and the immunological response of T cells was assessed (Fig.1A). Although transient lymphopenia was observed after neutron
irradiation, the number of immune cells was restored at
11 days after treatment. Assessing IFN- production, theantigen specific immune response was comparable inneutron irradiated mice, compared with control mice (Fig.
1B), suggesting that antigen-specific immune response
could be induced even after neutron irradiation.
Anti-tumor immune response after BNCT
It was previously reported that local X ray radiotherapy lead to the augmentation of anti-tumor immune response via the induction of inflammation and immunological cell death in tumor site [5]. Hence, we examineanti-tumor immune response in hosts which received
BNCT or X ray radiotherapy. Mice were challenged
B16 melanoma cells on leg, and, BNCT or local X ray
irradiation (12Gy) therapy were performed around 10
days after tumor challenge. Fourteen days after radiation
therapy, immune response against tumor derived antigen
was assessed (Fig.2 A). As a result, similar to previous
reports, anti-tumor immune response was augmented byradiotherapy with X ray (data not shown).

REFERENCES:[1] M. Suzuki, et al. Int. J. Radiat. Oncol. Biol.
Phys. 58(3): 892-896, 2004.
[2] M. Suzuki, et al. Int. J. Radiat. Oncol. Biol.
Phys. 60(3): 920-927, 2004.
[3] Lifu Dengu et al., J. C. I., 124 (2), 687, 2014.
[4] M. A. Curran et al., PNAS, 107(9), 4275, 2010.
[5] Lionel A. et al., Nature Med., 13(9), 1050,2007

採択課題番号 26049 BNCT の免疫に与える影響と免疫療法による全身治療との併用の可能性 通常採択
東理大・生命研）安部 良、鈴木利宙、小川修平、若松 英、堀江和峰（京大・原子炉）櫻井良憲、
小野公二

128

CO6-4In situ Visualization of Boron in Plants Using Neutron Capture Radiography

M. Kobayashi and T. Kinouchi1

part and a circular region just inside the cortex, which are
the regions rich in vascular bundles. Boron as borate
diester is specifically bound to the rhamnogalacturonan II(RG-II) regions of pectin in cell wall, and the RG-II has
been shown to occur in all cells of the roots at the samedensity [3]. Thus the distribution of B within roots seems
distinct from that of its acceptor molecule.
Besides borate diesters bound to RG-II regions ofpectin, B in plants can occur as free boric acid as well,when the plants are amply fed with B [4]. The free boric
acid in shoots corresponds to B taken up as luxury absorption beyond the amount equivalent to that of the acceptor. Radish seedlings used in this study were grown in
the presence of enough amount of B, and hence they werelikely to contain both RG-II-bound and free boric acid.
At present, we are not sure to which fraction of B thesignals in radiography are ascribed. However, the observed pattern of pit distribution seems inconsistent withthose expected for bound B: since the acceptor RG-II
occurs ubiquitously and evenly within the root, the bound
B is also expected to occur uniformly. Taken together, we
think that the signal detected in this study probably camefrom soluble, free boric acid in root tissues.
Radish deficient in B sometimes accumulates a blue
pigment in the roots, which is called “Ao-aza” (blue spot)
symptom. Interestingly, the regions where the blue pigment (presumably anthocyanin) accumulates overlap withthose showing signals in this study. It is suggested thatcells in the regions may require higher supply of B thanthe others. In the vascular bundles occurs cambium,
which contains the cells actively propagating. Higher
supply of B may be necessary to support rapid cell proliferation in these tissues.
As the subject of future investigation, it is necessary
to make clear which fraction of B (cell wall-bound or freeboric acid) has been detected in this radiography. Growing plants under various B supply to prepare samples
with different content of free boric acid, then examining
the pit frequency would give useful information on this
aspect. In addition, increasing the sensitivity and resolution is also necessary. Optimizing the condition of neutron irradiation or alkaline etching would be useful tosolve these problems.

Graduate School of Agriculture, Kyoto University
Research Reactor Institute, Kyoto University

1

INTRODUCTION:
Boron (B) is an essential micronutrient for vascularplants, and its deficiency is one of the major constraints
on crop production worldwide [1]. Boron deficiency
causes various physiological disorders including an inhibition of the development of young leaves, necrosis oftissues inside tubers or tap roots, or failure in seed set.
The understanding of physiological function of B inplants has progressed greatly during the last few decades.
Boron as boric acid occurs in cell wall, and forms 1:2
borate-diol diester with specific regions of pectin [2]. The
diester bonds serve as the inter-chain bridges of pectin,
thereby stabilizes the cell wall structure. On the other
hand, the transport and distribution of B in plant remains
elusive, since no radioactive isotope with an appropriate
half-life is available for this element. Revealing a differential distribution of B within tissues, if any, may give us
clues to the demands for B of different cell types, hence
may contribute significantly to a better understanding of
the physiological role of B. Thus, in this study we havebeen trying to apply boron neutron capture radiography
to the in situ visualization of B in plant roots.
EXPERIMENTS:
Radish (Raphanus sativus L. sativus) seeds were germinated on vermiculite in a growth chamber with alight/dark regime of 18/6 h, 22°C, and 60% relative humidity. Seedlings with uniform size were transferred tosolution culture, which was maintained in the same
growth chamber. The nutrient solution contained B at
0.5ppm. At 10 days after transfer to solution culture, thetap roots were detached from the seedlings and fixed withglutaraldehyde. Fixed samples were embedded in OCT
compound (Tissue-Tek), frozen in liquid nitrogen, andsectioned at 8-µm thickness with freezing microtome.
The ultrathin sections were transferred onto CR-39 nuclear track detector. The bright-field image of the section
was taken at this point, to record the structure of tissue
examined. The CR-39 plate was then irradiated with neutron for 15 min using Tc-Pn facility at Kyoto University
Research Reactor Institute. The irradiated CR-39 plate
was etched in NaOH solution, and the resulting pits wereobserved under microscope.

REFERENCES:[1] V.M. Shorrocks, Plant Soil, 193 (1997) 121–148.
[2] M. Kobayashi et al., Plant Physiol., 110 (1996)
1017–1020.
[3] T. Matoh et al., Plant Cell Physiol., 39 (1998)
483–491.
[4] T. Matoh et al., Soil Sci. Plant Nutr., 47 (2001)
779–784.

RESULTS AND DISCUSSION:
When the image of pits generated by neutron irradiation was merged with the section image taken prior to theirradiation, the pits were distributed unevenly over thesection. Higher density of pits was found in the central	
 採択課題番号 26056	
 	 	
 中性子捕捉反応を利用した植物組織におけるホウ素動態の解析	 	  通常採択	 （京大・農）小林	  優（京大・原子炉）木野内忠稔	
 
	
 
129

CO6-5

Measurement of Transmittance Spectra of a Cryo-Sectioned Tissueof Brain Tumor C6 Model in the Sub-Terahertz Reagion-II

N. Miyoshi and T. Takahashi1
Department of Tumor Pathology, Faculty of Medicine,
University of Fukui
1
Research Reactor Institute, Kyoto University

A

INTRODUCTION: The LINAC (linear particle accelerator) technology in the millimeter and terahertz waves
had been unique and had been used as a coherent synchrotron light in the research reactor institute of Kyoto
university (KURRI) to observe the transmittance spectraof a sectioned tissue of raw brain tumor C6 model as acollaborate study. The absorption spectra in the
sub-terahertz region had been not so clear for the raw
tumor tissue although Ashworth-PC. et al. [1] had reported for the excised human breast cancer by a terahertz
pulsed spectroscopy observed at 320 GHz, which wasestimated a longer relaxation time component of the induced electricity for water molecules [2-3] in the raw
tumor tissue for three years at the linear analysis.
We also estimated what kind of water molecules become
dominant in the viable and necrotic cancer regions by thedifferent measurement method as an aim of 2D mapping
study.

(2) Sample preparation: A cryo-sectioned (thickness=100m) tissue was prepared from the raw C6 glial tumor
model using a Cryo-section Maker (Leica) and wassealed sandwich-type with saran-wrap film (thickness=10m), under freezing condition (-20 C) before the measurements.
RESULTS: Large amount of spectroscopic data (morethan 1,000) for the 2D mapping data have been analyzed
to now, but the analysis was not finished to be reported as
the imaging of each water components.
REFERENCES:[1] Phillip C. Ashuworth, et. al., Optics Express, 17(14):12444-12454 (2009).
[2] Toshiko Fukasawa, et al., Phys. Rev. Let., 95: 197802(2005).
[3] Hiroyuki Yada, et al., Chem. Phys. Let., 464: 166-170(2008).

EXPERIMENTS: (1) Instrument of Near-field intera-hertz region: The photograph of the instrument was
shown in Figure 1. Mark-A: Pre-probe Wiston cone;50-10mm diameter, Length= 60mm; the irradiate diameter= 0.775mm; The concentrate light probe (diameter)= 3mm. The instrument was developed by Dr. T.
Takahashi in KURRI [Figure-1] for the 2D mapping
analysis.

採択課題番号 26072

テラヘルツ近接場分光装置を用いた実験脳腫瘍モデル組織のイメージング分光（福井大・医）三好憲雄（京大・原子炉）高橋俊晴
130

通常採択

CO6-6

Design and Synthesis of New Drugs for Boron Neutron Capture Therapy andBoron Magnetic Resonance Imaging

S. Aoki1, Y. Nishiura1, T. Tanaka1, Y. Hisamatsu1, Y.
Sawamoto1, Rikita Araki2, Takaomi Saido3, T. Suzuki4, K.
Horie4, Ryo Abe4, S. Masunaga5, K. Natsuko5, Y. Sakurai5, and K. Ono5

[3].
These results prompted us to design and synthesize its
analog (3) having an icosahedral carborane (o-carborane)
unit that consist 10 borons and 2 carbons as a potent carrier of boron cluster to the cancer cells. It was found that
this molecule is transferred into cancer cells more effectively than 1. Moreover, we have discovered that theo-carborane unit of 3 decomposes in the presence ofd-block metals in aqueous solution at neutral pH and release more than 4 molecules of B(OH)3 [4]. Besides,
o-carborane analog 4 having methanol unit instead ofcyclen also undergoes the full decomposition to release10 B(OH)3. This change was successfully detected on 11B
NMR and MRI. Because the chemical and physical properties of 10B and 11B are almost identical, these probes
should be useful not only for BNCT, but also for the determination of the distribution of boron-containing drugs
in the bodies and of the biorelevant d-block metals.

1

Faculty of Pharmaceutical Sciences, Tokyo University of
Science2
Bruker Biospin K. K.
3

RIKEN Brain Science Institute4

Research Institute of Biomedical Sciences, Tokyo University of Science
5
Research Reactor Institute, Kyoto University

INTRODUCTION: Boron neutron capture therapy(BNCT) is one of powerful therapies for local tumor control in the treatment of brain tumor, melanoma, and so on[1]. However, some important issues are remained for theachievement of successful tumor regression: the tumor-specific accumulation of highly concentrated boron,
the real-time quantification of boron concentration in
local tumor tissues, and the prevention of re-growth andmetastasis of residual tumor cells after BNCT. In thisstudy, we have designed and synthesized new boron
compounds appended with macrocyclic tetramines suchas cyclen (1,4,7,10-tetraaminocyclododecane) for BNCT
and B-MRI [2]. It was expected that boron clusters suchas icosahedral carboranes would be transferred to living
cells due to the dicationic properties of cyclen part at
neutral pH.
Icosahedral carboranes are boron clusters composedwith 10 boron atoms and 2 carbon atoms which exhibit aremarkable thermal and chemical stability. In this maniscript, we reported first example of full deboranation reaction of o-carborane catalyzed by copper(II) ion. Initially,
we examined the deboronation reaction of ortho-carborane-cyclen in aqueous solution in the presenceof various d-block metals such as Cu2+, Zn2+, Fe2+, Ni2+
and Co2+.

Fig 1. Design and decomposition reaction ofboronylated compounds
REFERENCES:[1] a) R. F. Barth et al., Clin. Cancer Res., 11 (2005)
3987-4002. b) R. F. Barth et al. Rad. Oncol. 7 (2012)
146-166.
[2] a) G. W. Kalbalka, et al. J. Neuro-Oncol. 33 (1997)
153-161. b) P. Bendel, NMR in Biomed. 18 (2005) 74-82.
[3] M. Kitamura et al., Inorg. Chem. 50 (2011)
11568-11580.
[4] Y. Nishiura et al., Manuscript in preparation.

EXPERIMENTS and RESULTS:
Synthesis of boron compound based on
Boron is one of ultratrace elements in living systems.
Previously, we have designed and synthesized boron-pendant cyclic tetraamine 1 for the in-cell 11B NMR(nuclear magnetic resonance) or MRI (magnetic resonance imaging) for the detection of metal in living systems and eventually for the use in BNCT. It was reported
that the C-B bond is hydrolyzed upon complexation withd-block metals to giveone molecule of boric acid
(B(OH)3) as well as zinc complex of N-benzylcyclen 2

がん組織に集積する含ホウ素化合物の設計・合成と
共同通常
通常採択
腫瘍への集積のホウ素 MRI に向けた基礎検討（東理大・薬学部）青木 伸、西浦由紀子、田中智博、久松洋介、澤本泰宏（ブルカーバイオスピン）荒木力太（理研）西道 隆臣（東理大・生命研）鈴木利宙、堀江和峰、
安部良（京大・原子炉）増永慎一郎、近藤夏子、櫻井良憲、小野公二
採択課題番号 26077

131